Frizzled 2 as a target for therapeutic antibodies in the treatment of cancer

ABSTRACT

Disclosed herein are methods of treating cancer in a subject, and methods for inhibiting growth, migration and/or invasion of a cancer cell in the subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen binding fragment thereof that downmodulates Fzd2. The antibody may specifically bind Fzd2, and may promote internalization of the Fzd2 receptor by the cancer cells and/or prevent ligand binding to Fzd2. Specific antibodies, and also specific portions of the Fzd2 molecule for antibody binding are disclosed. In one embodiment the antibody specifically binds to the epitope HGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1). Specific cancers (e.g. late stage hepatocellular carcinoma), intended for treatment are provided, and include cancers that exhibit overexpression of Fzd2, and/or Wnt5a.

RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 14/127,017 filed May 13, 2014, which is a 35 U.S.C. §371 NationalPhase Entry Application of International Application No.PCT/US2012/0042770 filed Jun. 15, 2012, which designates the U.S., andwhich claims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication Ser. No. 61/498,353 filed Jun. 17, 2011, the contents ofeach of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 8, 2016, isnamed “Sequence_Listing_002806-069427-C” and is 6,029 bytes in size.

GOVERNMENTAL SUPPORT

This invention was made with Government support under GM072872 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to the field of cancer therapeutics.

BACKGROUND OF THE INVENTION

Liver cancer is the third most common cause of cancer mortality, withapproximately 500,000 to 1 million annual deaths worldwide [1]. TheAmerican Cancer Society estimated that about 24,000 new cases of livercancer occurred in the US in 2010, with more than 80% of these beinghepatocellular carcinoma (HCC). An estimated 19,000 deaths in the US in2010 resulted from liver cancer. The incidence and death rates for livercancer have continued to increase since the early 1980s.

To date, surgical resection is considered the best treatment for livercancer, but only a small proportion of patients qualify for surgery andthere is a high rate of recurrence [2]. Many patients with HCC do notreceive any therapy. Liver transplantation has been successful intreating limited-stage HCC. Only a minority of patients with HCC,however, qualifies for transplantation. Standard chemotherapy is poorlytolerated in patients who do not qualify for resection. Both doxorubicinand cisplatin are frequently used, but overall response rates are low,and neither prolongs survival substantially. The 5-year survival ratefor patients with liver cancer is 14%. Five-year survival is 26% amongpatients in whom cancer is found at an early stage, compared to only 2%when it is found after spreading to distant organs. It is thereforecritical to develop a better understanding of hepatocarcinogenesis atthe molecular level to identify novel therapeutic targets that may playa pivotal role in the pathogenesis of this devastating disease.

HCC is the most common tumor that originates in the liver [2]. Recentstudies have highlighted the role of various signaling pathways in livercarcinogenesis, including the Wnt/β-catenin pathway, Hedgehog signaling,and receptor tyrosine kinase-related pathways. These discoveries offerpotential alternatives for novel targeted therapeutics. In particular,the role of Wnt signaling has been the subject of considerable interestin understanding the molecular pathogenesis of HCC [3]. Aberrant Wntsignaling has been implicated in many types of cancer, including cancersof the colon, skin, brain, liver and prostate [4]. In colorectalcarcinomas, abnormal accumulation of β-catenin arises primarily as aresult of mutations in β-catenin itself, as well as in APC and axin.These mutations are relatively rare in HCC, however, suggesting thatmisregulation of the Wnt pathway arises in other ways, includingoverexpression of other components of the pathway, such as Wnt ligandsand Fzd receptors [3]. To date, however, it is not known which ligandsor receptors are responsible for activation of the β-catenin pathway inHCC.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a method of treating cancer in asubject comprising administering to the subject a therapeuticallyeffective amount of an antibody or antigen binding fragment thereof thatdownmodulates Fzd2, such that the antibody or antigen binding fragmentthereof is delivered to cancer cells of the subject, to thereby treatthe cancer.

One aspect of the invention relates to a method of inhibiting growth,migration and/or invasion of a cancer cell in a subject comprisingadministering to the subject a therapeutically effective amount of anantibody or antigen binding fragment thereof, that downmodulates Fzd2,such that the antibody or antigen binding fragment thereof is deliveredto the cancer cells, to thereby treat the cancer.

In one embodiment of the various methods described herein the antibodyspecifically binds Fzd2. In one embodiment of the various methodsdescribed herein the antibody binds to Fzd2 and promotes internalizationof the Fzd2 receptor by the cancer cells. In one embodiment of thevarious methods described herein the antibody to Fzd2 prevents ligandbinding to Fzd2. In one embodiment of the various methods describedherein the antibody specifically binds an extracellular portion of theFzd-2 protein. In one embodiment of the various methods described hereinthe antibody specifically binds to Fzd2 within a region of Fzd2corresponding to amino acids 24-247 of Fzd2. In one embodiment of thevarious methods described herein the antibody specifically binds to Fzd2within a region of Fzd2 corresponding to amino acids 125-163 of Fzd2. Inone embodiment of the various methods described herein the antibodyspecifically binds to Fzd2 within a region of Fzd2 corresponding toamino acids 134-163 of Fzd2. In one embodiment of the various methodsdescribed herein the antibody specifically binds to Fzd2 within a regionof Fzd2 corresponding to amino acids 144-163 of Fzd2. In one embodimentof the various methods described herein the antibody specifically bindsto the epitope HGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1).

In one embodiment of the various methods described herein the antibodyis monoclonal. In one embodiment of the various methods described hereinthe antibody is polyclonal. In one embodiment of the various methodsdescribed herein the antibody is humanized.

In one embodiment of the various methods described herein the cancer isselected from the group consisting of gastrointestinal cancer, prostatecancer, ovarian cancer, breast cancer, head and neck cancer, lungcancer, non-small cell lung cancer, cancer of the nervous system, kidneycancer, retina cancer, skin cancer, liver cancer, pancreatic cancer,genital-urinary cancer and bladder cancer. In one embodiment of thevarious inventions described herein the cancer is liver cancer. In oneembodiment of the various inventions described herein the cancer is latestage hepatocellular carcinoma. In one embodiment of the various methodsdescribed herein the cancer displays overexpression of Fzd2. In oneembodiment of the various methods described herein the cancer displaysoverexpression of Wnt5a.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1B show experimental results which indicate Fzd2 isoverexpressed in late stage and poorly differentiated Hepatocellularcarcinoma (HCC). 48 tissue samples obtained from patients withhistopathologically confirmed HCC (stage I, n=7; stage II, n=8; stageIII, n=8; stage IV, n=3; tumor lesion, n=14), as well as in normal liversamples (n=8). FIG. 1A is a bar graph showing Fzd2 mRNA expression wassignificantly increased in late stages (Stage III and IV) ofhepatocellular carcinoma compared with normal tissue (P<0.05). FIG. 1Bis a bar graph showing the mRNA levels of Fzd2 expression correlatedwith tissue differentiation. Moderately and poorly differentiated tumorsshowed higher levels of Fzd2 compared with well differentiate tumortypes.

FIG. 2 shows experimental results which indicate Fzd2 is highlyoverexpressed in FOCUS cell line compared with more epitheliallike HepG2HCC cell line. A bar graph showing relative mRNA expression of variousWnt signaling receptors and co-receptors in FOCUS cells compared withHepG2 cells.

FIG. 3A-FIG. 3B show experimental results which indicate Fzd2 plays anessential role in Wnt5a-mediated cellular migration and invasion. FIG.3A and FIG. 3B are bar graphs showing siRNA-medicated knockdown of Fzd2or pre-treatment with anti-fzd2 antibody reduces cellular migration(FIG. 3A) and invasion (FIG. 3B) effects of Wnt5a at 2 hours.

FIG. 4A-FIG. 4B show experimental results which indicate. Anti-Fzd2antibody causes internalization of cell surface Fzd2 receptors. FIG. 4Ais a photograph of Western blots of biotinylated cell surface Fzd2protein shows reduced protein levels upon Wnt5a or anti-Fzd2pre-treatment. FIG. 4B is a bar graph showing relative quantitation ofcell surface Fzd2 levels normalized for total Fzd2 in whole celllysates.

FIG. 5 shows experimental results which indicate Fzd2 knockdown reducestumor growth in nude mice. FOCUS cells were injected s.c. into athymicmice and the ability of cells to form tumor outgrowths was monitored inthe presence or absence of siRNA against Fzd2. A tumor growth curve fortreated and control group showing in the presence of siRNA directedagainst Fzd2, tumor growth was significantly slower with a notable lagexponential growth phase is shown. Animals were injected with Fzd2-siRNAor Control.

FIG. 6A-FIG. 6B show experimental results which indicate Epitope mappingof anti-Fzd2 antibody. FIG. 6A, left, is a schematic showing full lengthdomain structure of Fzd2 protein. The domain structures of threefragments designed to map the epitope of anti-Fzd2 antibody are alsoshown. FIG. 6A, right, is a photograph of a Western blot showinganti-Fzd2 antibody can recognize only fragments 1 and 3. FIG. 6B, left,is a schematic showing the positions of overlapping residues in threepeptides designed to map the epitope of anti-Fzd2 antibody. FIG. 6B,right, is a peptide array image showing anti-Fzd2 antibody recognizesonly Peptide 2. Whole cell lysates from HEK293 cells overexpressing fulllength Fzd2 gene or empty vector (negative) were also printed on thesearrays. CRD, cystein rich domain; TM, transmembrane.

FIG. 7 is the amino acid sequence of the human Fzd2 protein (SEQ ID NO:2).

FIG. 8A-FIG. 8F show experimental results which indicate Fzd2 plays anessential role in Wnt5a-mediated cellular migration, invasion and tumorgrowth. FIG. 8A, left, is a bar graph showing relative mRNA expressionof five Fzd2-shRNA or control-shRNA expression FOCUS cells. FIG. 8A,right, is an image of western blots showing knockdown of Fzd2 proteinlevels. FIG. 8B is a graphical representation of data that showsknockdown of Fzd2 reduces FOCUS cell migration. Relative wound densityof parental (wt) or Fzd2-shRNA or control-shRNA expressing FOCUS cellsmonitored for 60 h. FIG. 8C, left, is a bar graph of data showing FOCUScells, in the presence of exogenous Wnt5a in the bottom of a Boydenchamber, treated with or without anti-fzd2 antibody reduces cellularmigration at 2 h. FIG. 8C, right, is a bar graph of data showing FOCUScells, in the presence of exogenous Wnt5a in the bottom of a Boydenchamber, treated with or without anti-fzd2 antibody reduces cellularinvasion. FIG. 8D is a collection of data indicating anti-Fzd2 antibodycauses internalization of cell surface Fzd2 receptors. FIG. 8D, left,are images of Western blots of biotinylated cell surface Fzd2 protein.Reduced protein levels were observed upon Wnt5a or anti-Fzd2pre-treatment. FIG. 8D, right, is a bar graph showing relativequantitation of cell surface Fzd2 levels normalized for total Fzd2 inwhole cell lysates. FIG. 8E and FIG. 8F are graphical representations ofdata that indicate Fzd2 knockdown reduces tumor growth in nude mice.FOCUS cells were injected s.c. into athymic mice and the ability ofcells to form tumor outgrowths was monitored in the presence or absenceof siRNA against Fzd2 (FIG. 8E) or Fzd2-shRNA (FIG. 8F).

FIG. 9 shows Kaplan-Meier survival curves for 40 HCC patients. Thestatistical p value was generated by the Cox-Mantel log-rank test.

FIG. 10 is a graphical representation of binding analysis studiesperformed with the anti-fzd2 antibody (BioCore study performed byPrecision Antibody™). The analysis indicated that the KD of thecommercial Fzd2 Ab used in the experiments herein against Fzd2 is ˜20nM.

FIG. 11A-FIG. 11B show experimental results that indicate Fzd2 mediatesrelease of SerpinE1 and sICAM1. FIG. 11A contains images of humancytokine array probed with conditioned media from FOCUS-WT (top) andFOCUS-shFzd2 (bottom). After detection, the array data were quantifiedto generate a protein profile. FIG. 11B is a bar graph showing abundanceof 36 cytokines measured using this assay. The amount of SerpinE1 andsiCAM1 released was significantly lower in the condition media fromFOCUS-shFzd2 cells.

FIG. 12 shows experimental results that indicate matrixmetalloproteinases (MMPs) and SerpinE1 are overexpressed in late stage,aggressive and poorly differentiated Hepatocellularcarcinoma (HCC)lines. A bar graph shows relative mRNA expression of various MMPs andSerpinE1 in early stage nonaggressive (HepG2 and Huh7) and late stage,aggressive (SNU475 and FOCUS) cell lines. mRNA expression was normalizedto 18S.

FIG. 13 shows experimental results that indicate Fzd2 regulates the mRNAexpression of matrix metalloproteinases (MMPs) and SerpinE1. A bar graphshows relative mRNA expression of various MMPs and SerpinE1 in FOCUS-CTLor FOCUS-shFzd2 cells.

FIG. 14A-FIG. 14C shows experimental results that indicate Fzd2regulates the phosphorylation status of Stat3, ERK1/2 and MEK1/2 as wellas its transcription activity. FIG. 14A is an image of an Immunoblotshowing STAT3, ERK1/2, and MEK1/2 phosphorylation in wild-type FOCUScells, and FOCUS cells with knockdown of Fzd2. Similar results weredemonstrated in other late stage cell lines and with treatment withanti-Fzd2 antibody. FIG. 14B is a bar graph showing stat3 transcriptionactivity using a reporter/luciferase-based assay in wild-type FOCUS andSNU449 cells, and cells knockdown with Fzd2 or STAT3. Similar resultswere demonstrated in other late stage cell lines and with treatment withanti-Fzd2 antibody. FIG. 14C is a collection of dose-response curvesshowing the effect of small molecule inhibitor against Stat3 on cellmigration of late stage HCC cell lines (FOCUS).

FIG. 15A-FIG. 15C shows experimental results that indicate Fzd2regulates the phosphorylation status of Src family kinases whichphosphorylate Stat3 in late stage HCC cell lines. FIG. 15A is a bargraph showing phosphorylation of src family kinases in wild-type FOCUScells, and FOCUS cells with knockdown of Fzd2. Similar results weredemonstrated in other late stage cell lines. FIG. 15B is a photograph ofexperimental results that indicate the molecule inhibitor (dasatinib)against SFK abolishes stat3 phosphorylation in FOCUS cells. FIG. 15C isa collection of dose-response curves showing the effect of smallmolecule inhibitor against SFK on cell migration of late stage HCC celllines (FOCUS).

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the invention relate to the finding that specific binding ofthe Frizzled 2 receptor (Fzd2) on hepatocellular carcinoma cells by anantibody reduces various tumorigenic properties of the cells. Theantibody binding reduces the cell migration, the cell invasion, as wellas growth of the cells. As such, one aspect of the invention relates toa method for inhibiting one or more tumorigenic properties of a tumorcell (e.g., growth, migration and/or invasiveness). The method comprisescontacting the tumor cell with an effective amount of an agent thatdownmodulates Fzd2 in the cell (e.g. an antibody or antigen bindingfragment thereof), to thereby downmodulate the Fzd2 in the cell. In oneembodiment, the agent specifically downmodulates the Fzd2 in the cell,that is to say the agent does not downmodulate other closely relatedproteins (e.g., other Fzd proteins described herein). In one embodiment,the agent promotes sufficient internalization of the Fzd2 receptor bythe cancer cells to inhibit one or more tumorigenic properties of thecell. In one embodiment, the agent prevents ligand (Wnt5a) binding toFzd2.

The findings reported herein can be extrapolated to the treatment of atumor (e.g., cancer) in a subject. As such, another aspect of theinvention relates to a method of treating a tumor (e.g., a cancer) in asubject. The method comprises administering to the subject atherapeutically effective amount of an agent (e.g., an antibody orantigen binding fragment thereof) that downmodulates Fzd2. In oneembodiment, the agent specifically downmodulates the Fzd2 in the cell.Administration is performed by a method that delivers an effectiveamount of the agent to a sufficient portion of the tumor cells of thesubject to thereby produce therapeutic results. Effective treatmentresults in inhibition of one or more properties of the tumor cells,including growth, migration, and invasion, of the tumor cells in thesubject. In one embodiment, the treatment is useful for inhibition ofmetastasis of the tumor cells in the subject.

The methods described herein are applicable to the treatment of tumorsat various stages of disease progression, ranging from preliminarystages of disease to significantly advanced stages of disease. Theadvanced stage of disease wherein the tumor exhibits the properties ofhigh growth rate, significant migration and invasiveness, is oftensignified by the detection of metastasis of the tumor cells to otherlocations or organs in the subject (e.g, lymph nodes, lungs, brain). Inone embodiment, one or more of the properties of loss of growth control,migration, and invasiveness are exhibited by the tumor.

It may be advantageous to determine the rate of the tumor cell targetedby the methods described herein prior to treatment. A reproducible,statistically significant amount as detected by standard means in theart is sufficient to indicate the presence of the one or more propertiesof the tumor cells described herein. The extent of each property (e.g.,rate of growth and degree of migration and invasive properties) canfurther be measured by standard methods in the art. Such methods can beused to determine the state of the tumor cell with respect to theseproperties, following or in the course of treatment, to detectinhibition (reduction of the one or more properties) of the tumor cellsby the treatment.

In one embodiment, one or more of the properties (growth, migration,invasion, and metastasis) are undetectable in the tumor cells of thesubject. In such a situation, the method described herein may result indelay or prevention of onset of one or more of the properties.

Target Cell Types

The methods described herein are suitable for use on a variety of tumorcell types. The tumors may be primary tumors or may be secondary tumors.The tumors may originate from any cell or organ in a subject. In oneembodiment, the tumor is an epithelial tumor. In one embodiment thetumor arises in the gastrointestinal tract, prostate, ovary, breast,cervix, central nervous system, peripheral nervous system, lung, kidney,retina, blood, skin, liver, pancreas, or the genital-urinary system(e.g., testes, bladder) of a subject.

The tumor may have progressed to the stage of cancer. In one embodiment,the cancer is gastrointestinal cancer, prostate cancer, ovarian cancer,breast cancer, head and neck cancer, lung cancer, lymphoma, non-smallcell lung cancer, cancer of the nervous system, kidney cancer, retinacancer, skin cancer (e.g., basal cell carcinoma, squamous cellcarcinoma, and melanoma), liver cancer, pancreatic cancer,genital-urinary cancer or bladder cancer. In one embodiment, the cancercell is a carcinoma, sarcoma, lymphoma, germ cell tumor or blastoma. Inone embodiment, the cancer is hepatocellular carcinoma.

The cancer can be in any possible stage of disease progression (e.g.,stage 0, I, II, III, IV). In one embodiment, the cancer is late stagehepatocellular carcinoma. In one embodiment, the cancer has exhibitedsigns of metastasis.

In one embodiment, the tumor exhibits overexpression of Fzd2 and/orWnt5a, as compared to an appropriate control cell or tissue.Overexpression refers to an increased amount or level of the protein.Such an increase is detected as an identified, reproducible,quantitative or qualitative increase in the physical presence of atarget molecule or molecules (e.g., Fzd2 or Wnt5a). An increase that isconsidered relevant to the methods described herein is an increase by atleast about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about100% of the protein level in an appropriate control. Significantlyhigher increases above a 2 fold increase would also be relevant to themethods described herein. Additionally, overexpression of a targetprotein can be determined by analysis of the mRNA in the cell encodingthe target protein. An increase that is considered relevant to themethods described herein is an increase by at least about 5%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 95%, about 99%, about 100% of the mRNA level in anappropriate control. Significantly higher increases above a 2 foldincrease would also be relevant to the methods described herein.

An increased amount is determined by measuring (e.g., quantitative) thetarget molecule(s) in a target cell or tissue, to produce a determinedamount, followed by comparison to a control amount obtained by measuringthe target molecule(s) in an appropriate control cell or tissue, undernormal or non-disease conditions. Measurement in the target cell ortissue and in the control cell or tissue is performed by as close toidentical methods as possible under the given experimental conditions.An appropriate control cell can be determined by the skilledpractitioner. For example, normal adjacent tissue of the same cell typeis often used as a control.

Downmodulation of the Fzd2 protein that results from the methodsdescribed herein can be detected by a variety of means. Downmodulationresulting from decreased Fzd2 protein expression can be detected byquantitative measurement of the protein in a cell. Downmodulationresulting from reducing one or more protein functions (e.g., ligandbinding) can be detected by analysis of that function (e.g., measuringthe amount of bound ligand). In one embodiment, downmodulation resultsfrom increasing internalization of the Fzd2 receptor. In one embodiment,the internalization reduces the total amount of Fzd2 receptor on thecell surface. In one embodiment, the Fzd2 receptor on the surface of thecell is reduced by at least about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 99%, about 100% of the amount on an appropriate control cell.

Fzd Proteins

The terms “frizzled protein” or “frizzled receptor” refer to a family ofmammalian proteins. The Frizzled family comprises at least 10 mammaliangenes. Human Frizzled receptors include Fzd1, Fzd2, Fzd3, Fzd4, Fzd5,Fzd6, Fzd7, Fzd8, Fzd9 and Fzd10. The sequences of the different humanFrizzled receptors are publically available. The mammalian frizzledproteins share a number of common structural motifs. The N terminuslocated at the extracellular membrane surface is followed by a signalsequence, a domain of 120 amino acids with an invariant pattern of 10cysteine residues, and a highly divergent region of 40-100 largelyvariable hydrophilic amino acids. Putative hydrophobic segments formseven membrane-spanning helices linked by hydrophilic loops, ending withthe C terminus located at the intracellular face of the membrane. Thecysteine-rich domains (CRDs) and the transmembrane segments are stronglyconserved, suggesting a working model in which an extracellular CRD istethered by a variable linker region to a bundle of sevenmembrane-spanning-helices. Frizzled protein receptors are, therefore,involved in a dynamic model of transmembrane signal transductionanalogous to G-protein-coupled receptors with amino-terminal ligandbinding domains.

The amino acid sequence of human Fzd2 is set forth in FIG. 7. Aminoacids 1-23 are the signal peptide. Extra cellular domains of Fzd2 areencompassed by amino acids 24-247, 301-327, 392-414, and 483-519.

Wnt5a

Wnt5a (wingless-related MMTV integration site 5a) is a member of a largefamily of cysteine-rich growth factors. Proteins in this family arehighly conserved and naturally secreted. The Wnt5a protein binds tomembers of the Frizzled (Fzd) family of seven-transmembrane domainreceptors on the cell surface, including Fzd2, and this triggers aseries of intracellular events that ultimately regulate genetranscription. These intracellular events are grouped according to twoknown signaling pathways, the canonical Wnt/β-catenin pathway (He etal., Science 275:652-654, 1997; Toyofuku et al., J. Cell Biol.150:225-41, 2000; Kawakami et al., Cell 104:891-900, 2001) and theWnt/Ca++ pathway (Slusarski et al., Dev. Biol. 182:114-120, 1997; Kuhlet al., J. Biol. Chem. 275:12701-12711, 2000; and Kuhl et al., Mech.Dev. 106:61-76, 2001). Upregulation of gene expression of WNT5a has beenobserved in various human cancers (Lejeune et al., Clin. Cancer Res.1:215-222, 1995; Tozzo et al., Cancer Res. 55:3495-3499, 1995) and WNT5ahas recently been reported to facilitate cell invasion in humanmetastatic melanoma (Weeraratna et al., Cancer Cell 1:279-88, 2002).Determination of expression levels of Wnt5a in a cell, tissue or aspecific cancer is described in detail in U.S. Pat. No. 7,723,055.

Agents for Downmodulation

In one embodiment, the agent for downmodulation is specific for Fzd2.Specific, as the term is used herein in reference to downmodulation ofFzd2, refers to an absence of significant downmodulation of otherrelated proteins, such as other Frizzled proteins or receptors (e.g.,Fzd1, or Fzd3-Fzd10). Agents that specifically downmodulate Fzd2, and donot significantly downmodulate other Fzd proteins can be generated byroutine methods.

Agents for downmodulation, as described herein can be chemicals; smallmolecules; nucleic acid sequences; nucleic acid analogues; proteins;peptides; aptamers; antibodies; or fragments thereof. Many such agentsare known in the art and can be used in the present invention. Othersuch agents can be identified or generated for use in the presentinvention.

Such an agent can take the form of any entity that is normally absent(exogenous) or present at lower levels that those provided to thecell(s). Agents such as chemicals; small molecules; nucleic acidsequences; nucleic acid analogues; proteins; peptides; aptamers;antibodies, or antigen binding fragments thereof, can be identified orgenerated for use to downmodulate the Fzd2.

Agents in the form of a protein and/or peptide or fragment thereof canbe used to downmodulate Fzd2. Examples of useful proteins are mutatedproteins, genetically engineered proteins, peptides, synthetic peptides,recombinant proteins, chimeric proteins, antibodies, midibodies,minibodies, triabodies, humanized proteins, humanized antibodies,chimeric antibodies, modified proteins and fragments thereof. Agentsalso include antibodies (polyclonal or monoclonal), neutralizingantibodies, antibody fragments, peptides, proteins, peptide-mimetics,aptamers, hormones, small molecules, carbohydrates or variants thereofthat function to inactivate the nucleic acid and/or protein of the geneproducts identified herein, and those as yet unidentified Inhibitoryagents can also be a chemical, small molecule, chemical entity, nucleicacid sequences, nucleic acid analogues or protein or polypeptide oranalogue or fragment thereof.

The agent may function directly in the form in which it is administered.Alternatively, the agent can be modified or utilized intracellularly toproduce something which downmodulates the Fzd2, such as introduction ofa nucleic acid sequence into the cell and its transcription resulting inthe production of the nucleic acid and/or protein inhibitor of Fzd2within the cell. The agent can be made from any chemical, entity ormoiety, including without limitation synthetic and naturally-occurringnon-proteinaceous entities. In certain embodiments the agent is a smallmolecule having a chemical moiety. For example, chemical moietiesincluded unsubstituted or substituted alkyl, aromatic, or heterocyclylmoieties including macrolides, leptomycins and related natural productsor analogues thereof. Agents can be known to have a desired activityand/or property, or can be selected from a library of diverse compounds.

Antibodies and Antibody Binding Fragments

Antibodies that bind an extracellular region of Fzd2 are particularlysuited for use in the methods described herein. In one embodiment, theantibody specifically binds Fzd2, and does not appreciably bind relatedFzd molecules. Antibodies that bind specifically to Fzd2 (e.g., theextracellular region), and do not significantly bind to other Fzdproteins can be generated or otherwise identified by routine methods. Byway of example, epitopes that are specific to Fzd2 can be used togenerate or identify antibodies or antigen binding fragments thereof, bythe methods described herein. The location of such epitopes can bedetermined, for example, by comparison of the amino acid sequences ofthe Fzd2 protein, to the amino acid sequences of related Fzd familymembers, to thereby identify regions of dissimilarity. In oneembodiment, the antibody binds an extracellular portion of the Fzd2protein. Useful extracellular portions of the Fzd2 protein include,without limitation, a region of Fzd2 corresponding to amino acids24-247, amino acids 125-163, amino acids 134-163, and amino acids144-163. In one embodiment, the antibody or antigen binding fragmentbinds a fragment of Fzd2 comprising the amino acid sequenceHGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1). In one embodiment, the antibody orantigen binding fragment binds the epitope within the amino acidsequence HGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1). In one embodiment, theantibody or antigen binding fragment binds a subset of the amino acidswithin the amino acid sequence HGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1), whenpresent in the context of the Fzd2 protein (e.g., having the amino acidsequence shown in FIG. 7). In one embodiment, the antibody or antigenbinding fragment binds 19 or less consecutive amino acids ofHGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1), within the context of the wild typeFzd2 protein. In one embodiment, the antibody or antigen bindingfragment binds 18 or less, 17 or less, 16 or less, 15 or less, 14 orless, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 orless, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless consecutive amino acids of HGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1),within the context of the wild type Fzd2 protein.

Antibodies which were generated to Fzd2 from non-human species are alsoexpected to recognize human Fzd2 if generated to epitopes which lie inareas of amino acid identity (e.g., 100% identity) to human Fzd2.

Detection of specific binding of an antibody to a region or epitope ofFzd2 is exemplified herein. The ability of a region or epitope tocompetitively inhibit binding of the antibody to a longer region ofFzd2, or to full length Fzd2 can also be used to identify the region orepitope to which an antibody or antigen binding fragment binds.

In one embodiment, the antibody binds to Fzd2, wherein that bindingpromotes internalization of the Fzd2 receptor by the tumor cells. In oneembodiment, binding of the antibody to Fzd2 prevents or otherwisesignificantly reduces binding of Fzd2 to endogenous ligand. One suchligand is Wnt5a.

An antibody used in the present invention can be of any one of thevarious immunoglobulin isotypes. An antigen binding fragment of anantibody described herein is also useful in the methods describedherein, and includes, without limitation, the Fab, scFv, Fv, dAb, and Fdfragments. Preferred for human therapeutic use are high affinity murine,chimeric, human and humanized antibodies, and antigen binding fragmentsthereof, having potent in vivo activity.

Structurally, the simplest antibody (IgG) comprises four polypeptidechains, two heavy (H) chains and two light (L) chains inter-connected bydisulphide bonds. The light chains exist in two distinct forms calledkappa (κ) and lambda (λ). Each chain has a constant region (C) and avariable region (V). Each chain is organized into a series of domains.The light chains have two domains, corresponding to the C region and theother to the V region. The heavy chains have four domains, onecorresponding to the V region and three domains (1, 2 and 3) in the Cregion. The antibody has two arms (each arm being a Fab region), each ofwhich has a VL and a VH region associated with each other. It is thispair of V regions (VL and VH) that differ from one antibody to another(owing to amino acid sequence variations), and which together areresponsible for recognizing the antigen and providing an antigen bindingsite (ABS). In even more detail, each V region is made up from threecomplementarity determining regions (CDR) separated by four frameworkregions (FR). The CDR's are the most variable part of the variableregions, and they perform the critical antigen binding function. The CDRregions are derived from many potential germ line sequences via acomplex process involving recombination, mutation and selection.

Certain identifiable fragments of a whole antibody also retain theability to bind antigen. Such binding fragments are (i) the Fab fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragmentconsisting of the VH and CH1 domains; (iii) the Fv fragment consistingof the VL and VH domains of a single arm of an antibody, (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989) which consistsof a VH domain; (v) isolated CDR regions; and (vi) F(ab′)2 fragments, abivalent fragment comprising two Fab fragments linked by a disulphidebridge at the hinge region.

Although the two domains of the Fv fragment are coded for by separategenes, it is possible to make a synthetic linker that enables them to bemade as a single protein chain (known as single chain Fv (scFv); Bird,R. E. et al., Science 242, 423-426 (1988) Huston, J. S. et al., Proc.Natl. Acad. Sci., USA 85, 5879-5883 (1988)) by recombinant methods.These scFv fragments were assembled from genes from monoclonalantibodies that had been previously isolated. In this application, theapplicants describe a process to assemble scFv fragments from VH and VLdomains that are not part of an antibody that has been previouslyisolated.

Antibodies useful in the present invention can be in the form ofpolyclonal, monoclonal, chimeric, humanized, and recombinant antibodies.Antigen-binding fragments can be generated from an antibody by methodsknown to the skilled practitioner. Antibodies are readily raised inanimals such as rabbits or mice by immunization with the antigen.Immunized mice are particularly useful for providing sources of B cellsfor the manufacture of hybridomas, which in turn are cultured to producelarge quantities of monoclonal antibodies.

Antibodies provide high binding avidity and unique specificity to a widerange of target antigens and haptens. Monoclonal antibodies useful inthe practice of the present invention include whole antibody andfragments thereof and are generated in accordance with conventionaltechniques, such as hybridoma synthesis, recombinant DNA techniques andprotein synthesis.

Polyclonal antibodies, or fragments thereof, can be derived from anyspecies.

Useful monoclonal antibodies and fragments can be derived from anyspecies (including humans) or can be formed as chimeric proteins whichemploy sequences from more than one species. Human monoclonal antibodiesor “humanized” murine antibody are also used in accordance with thepresent invention. For example, murine monoclonal antibody can be“humanized” by genetically recombining the nucleotide sequence encodingthe murine Fv region (i.e., containing the antigen binding sites) or thecomplementarily determining regions thereof with the nucleotide sequenceencoding a human constant domain region and an Fc region. Humanizedtargeting moieties are recognized to decrease the immunoreactivity ofthe antibody or polypeptide in the host recipient, permitting anincrease in the half-life and a reduction the possibly of adverse immunereactions in a manner similar to that disclosed in European PatentApplication No. 0,411,893 A2. The murine monoclonal antibodies shouldpreferably be employed in humanized form. Antigen binding activity isdetermined by the sequences and conformation of the amino acids of thesix complementarily determining regions (CDRs) that are located (threeeach) on the light and heavy chains of the variable portion (Fv) of theantibody. The 25-kDa single-chain Fv (scFv) molecule, composed of avariable region (VL) of the light chain and a variable region (VH) ofthe heavy chain joined via a short peptide spacer sequence, is thesmallest antibody fragment developed to date. Techniques have beendeveloped to display scFv molecules on the surface of filamentous phagethat contain the gene for the scFv. scFv molecules with a broad range ofantigenic-specificities can be present in a single large pool ofscFv-phage library. Some examples of high affinity monoclonal antibodiesand chimeric derivatives thereof, useful in the methods of the presentinvention, are described in the European Patent Application EP 186,833;PCT Patent Application WO 92/16553; and U.S. Pat. No. 6,090,923.

Chimeric antibodies are immunoglobin molecules characterized by two ormore segments or portions derived from different animal species.Generally, the variable region of the chimeric antibody is derived froma non-human mammalian antibody, such as murine monoclonal antibody, andthe immunoglobin constant region is derived from a human immunoglobinmolecule. Preferably, both regions and the combination have lowimmunogenicity as routinely determined.

One limitation of scFv molecules is their monovalent interaction withtarget antigen. One of the easiest methods of improving the binding of ascFv to its target antigen is to increase its functional affinitythrough the creation of a multimer. Association of identical scFvmolecules to form diabodies, triabodies and tetrabodies can comprise anumber of identical Fv modules. These reagents are thereforemultivalent, but monospecific. The association of two different scFvmolecules, each comprising a VH and VL domain derived from differentparent Ig will form a fully functional bispecific diabody. A uniqueapplication of bispecific scFvs is to bind two sites simultaneously onthe same target molecule via two (adjacent) surface epitopes. Thesereagents gain a significant avidity advantage over a single scFv or Fabfragments. A number of multivalent scFv-based structures has beenengineered, including for example, miniantibodies, dimericminiantibodies, minibodies, (scFv)2, diabodies and triabodies. Thesemolecules span a range of valence (two to four binding sites), size (50to 120 kDa), flexibility and ease of production. Single chain Fvantibody fragments (scFvs) are predominantly monomeric when the VH andVL domains are joined by, polypeptide linkers of at least 12 residues.The monomer scFv is thermodynamically stable with linkers of 12 and 25amino acids length under all conditions. The noncovalent diabody andtriabody molecules are easy to engineer and are produced by shorteningthe peptide linker that connects the variable heavy and variable lightchains of a single scFv molecule. The scFv dimers are joined byamphipathic helices that offer a high degree of flexibility and theminiantibody structure can be modified to create a dimeric bispecific(DiBi) miniantibody that contains two miniantibodies (four scFvmolecules) connected via a double helix. Gene-fused or disulfide bondedscFv dimers provide an intermediate degree of flexibility and aregenerated by straightforward cloning techniques adding a C-terminalGly4Cys sequence. scFv-CH3 minibodies are comprised of two scFvmolecules joined to an IgG CH3 domain either directly (LD minibody) orvia a very flexible hinge region (Flex minibody). With a molecularweight of approximately 80 kDa, these divalent constructs are capable ofsignificant binding to antigens. The Flex minibody exhibits impressivetumor localization in mice. Bi- and tri-specific multimers can be formedby association of different scFv molecules. Increase in functionalaffinity can be reached when Fab or single chain Fv antibody fragments(scFv) fragments are complexed into dimers, trimers or largeraggregates. The most important advantage of multivalent scFvs overmonovalent scFv and Fab fragments is the gain in functional bindingaffinity (avidity) to target antigens. High avidity requires that scFvmultimers are capable of binding simultaneously to separate targetantigens. The gain in functional affinity for scFv diabodies compared toscFv monomers is significant and is seen primarily in reduced off-rates,which result from multiple binding to two or more target antigens and torebinding when one Fv dissociates. When such scFv molecules associateinto multimers, they can be designed with either high avidity to asingle target antigen or with multiple specificities to different targetantigens. Multiple binding to antigens is dependent on correct alignmentand orientation in the Fv modules. For full avidity in multivalent scFvstarget, the antigen binding sites must point towards the same direction.If multiple binding is not sterically possible then apparent gains infunctional affinity are likely to be due the effect of increasedrebinding, which is dependent on diffusion rates and antigenconcentration. Antibodies conjugated with moieties that improve theirproperties are also contemplated for the instant invention. For example,antibody conjugates with PEG that increases their half-life in vivo canbe used for the present invention. Immune libraries are prepared bysubjecting the genes encoding variable antibody fragments from the Blymphocytes of naive or immunized animals or patients to PCRamplification. Combinations of oligonucleotides which are specific forimmunoglobulin genes or for the immunoglobulin gene families are used.Immunoglobulin germ line genes can be used to prepare semisyntheticantibody repertoires, with the complementarity-determining region of thevariable fragments being amplified by PCR using degenerate primers.These single-pot libraries have the advantage that antibody fragmentsagainst a large number of antigens can be isolated from one singlelibrary. The phage-display technique can be used to increase theaffinity of antibody fragments, with new libraries being prepared fromalready existing antibody fragments by random, codon-based orsite-directed mutagenesis, by shuffling the chains of individual domainswith those of fragments from naive repertoires or by using bacterialmutator strains.

In one embodiment, the antibody or antigen binding fragment thereof, isproduced by a SCID-hu mouse, for example the model developed byGenpharm. In one embodiment, a type of high avidity binding molecule,termed peptabody, created by harnessing the effect of multivalentinteraction is contemplated.

Human antibodies against Fzd2 can be produced from non-human transgenicmammals having transgenes encoding at least a segment of the humanimmunoglobulin locus. Usually, the endogenous immunoglobulin locus ofsuch transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/12227 (1993);U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat.No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553(1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741(1991) (each of which is incorporated by reference in its entirety forall purposes). Transgenic mice are particularly suitable. Anti-Fzd2antibodies are obtained by immunizing a transgenic nonhuman mammal, suchas described by Lonberg or Kucherlapati, supra with Fzd2 or a fragmentthereof. Monoclonal antibodies are prepared by, e.g., fusing B-cellsfrom such mammals to suitable myeloma cell lines using conventionalKohler-Milstein technology. Human polyclonal antibodies can also beprovided in the form of serum from humans immunized with an immunogenicagent Optionally, such polyclonal antibodies can be concentrated byaffinity purification using Fzd2 or other Fzd2 peptide as an affinityreagent.

A further approach for obtaining human Fzd2 antibodies is to screen aDNA library from human B cells according to the general protocoloutlined by Huse et al., Science 246:1275-1281 (1989). As described fortrioma methodology, such B cells can be obtained from a human immunizedwith Fzd2, fragments, longer polypeptides containing Fzd2 or fragmentsor anti-idiotypic antibodies. Optionally, such B cells are obtained froma patient who is ultimately to receive antibody treatment. Antibodiesbinding to Fzd2 or a fragment thereof are selected. Sequences encodingsuch antibodies (or a binding fragments) are then cloned and amplified.The protocol described by Huse is rendered more efficient in combinationwith phage-display technology. See, e.g., Dower et al., WO 91/17271 andMcCafferty et al., WO 92/01047, U.S. Pat. No. 5,877,218, U.S. Pat. No.5,871,907, U.S. Pat. No. 5,858,657, U.S. Pat. No. 5,837,242, U.S. Pat.No. 5,733,743 and U.S. Pat. No. 5,565,332 (each of which is incorporatedby reference in its entirety for all purposes). In these methods,libraries of phage are produced in which members display differentantibodies on their outer surfaces. Antibodies are usually displayed asFv or Fab fragments. Phage displaying antibodies with a desiredspecificity are selected by affinity enrichment to an Fzd2 peptide orfragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeU.S. Pat. No. 6,172,197, issued Jan. 9, 2001. In this method, either theheavy or light chain variable region of the selected murine antibody isused as a starting material. If, for example, a light chain variableregion is selected as the starting material, a phage library isconstructed in which members display the same light chain variableregion (i.e., the murine starting material) and a different heavy chainvariable region. The heavy chain variable regions are obtained from alibrary of rearranged human heavy chain variable regions. A phageshowing strong specific binding for Fzd2 (e.g., at least 108 andpreferably at least 109 M-1) is selected. The human heavy chain variableregion from this phage then serves as a starting material forconstructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions are obtained from alibrary of rearranged human variable light chain regions. Again, phageshowing strong specific binding for Fzd2 are selected. These phagedisplay the variable regions of completely human anti-Fzd2 antibodies.These antibodies usually have the same or similar epitope specificity asthe murine starting material.

In one embodiment, the agent inhibits gene expression (i.e., suppressand/or repress the expression of a gene of interest (e.g., the Fzd2gene)). Such agents are referred to in the art as “gene silencers” andare commonly known to those of ordinary skill in the art. Examplesinclude, but are not limited to a nucleic acid sequence, (e.g., for anRNA, DNA, or nucleic acid analogue). These can be single or doublestranded. They can encode a protein of interest, can be anoligonucleotide, a nucleic acid analogue. Included in the term “nucleicacid sequences” are general and/or specific inhibitors. Some knownnucleic acid analogs are peptide nucleic acid (PNA),pseudo-complementary PNA (pc-PNA), locked nucleic acids (LNA) andderivatives thereof. Nucleic acid sequence agents can also be nucleicacid sequences encoding proteins that act as transcriptional repressors,antisense molecules, ribozymes, small inhibitory nucleic acid sequences(e.g., RNAi, shRNAi, siRNA, micro RNAi (miRNA), and antisenseoligonucleotides. Many such molecules for inhibiting Fzd2 are known inthe art. As such these inhibitors can function as an agent in thepresent invention.

One type of downmodulatory agent for use in the present invention is anRNAi molecule (e.g., an siRNA or miRNA). The term “RNAi” and “RNAinterfering” with respect to an agent of the invention are usedinterchangeably herein. The term “RNAi” as used herein refers tointerfering RNA or RNA interference, which is a means of selectivepost-transcriptional gene silencing by destruction of specific mRNA bymolecules that bind and inhibit the processing of mRNA, for exampleinhibit mRNA translation or result in mRNA degradation. As used herein,the term “RNAi” refers to any type of interfering RNA, including but arenot limited to, siRNAi, shRNAi, endogenous microRNA and artificialmicroRNA. For instance, it includes sequences previously identified assiRNA, regardless of the mechanism of down-stream processing of the RNA(i.e. although siRNAs are believed to have a specific method of in vivoprocessing resulting in the cleavage of mRNA, such sequences can beincorporated into the vectors in the context of the flanking sequencesdescribed herein).

RNAi molecules are typically comprised of a sequence of nucleic acids ornucleic acid analogs, specific for a target gene. A nucleic acidsequence can be RNA or DNA, and can be single or double stranded, andcan be selected from a group comprising; nucleic acid encoding a proteinof interest, oligonucleotides, nucleic acid analogues, for examplepeptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), lockednucleic acid (LNA).

As used herein an “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a gene or target gene when the siRNA is present orexpressed in the same cell as the target gene, for example an HDF gene.The double stranded RNA siRNA can be formed by the complementarystrands. In one embodiment, a siRNA refers to a nucleic acid that canform a double stranded siRNA. The sequence of the siRNA can correspondto the full length target gene, or a subsequence thereof. Typically, thesiRNA is at least about 15-50 nucleotides in length (e.g., eachcomplementary sequence of the double stranded siRNA is about 15-50nucleotides in length, and the double stranded siRNA is about 15-50 basepairs in length, preferably about 19-30 base nucleotides, preferablyabout 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides in length). An siRNA can be chemicallysynthesized, it can be produced by in vitro transcription, or it can beproduced within a cell specifically utilized for such production.

In one embodiment, the siRNA is designed for inhibition of expression ofFzd2. Examples of such siRNA and methods of use to inhibit expressionare known in the art (Published Patent Application WO 2010/039679;Truong et al., Genes & Dev. 20: 3185-3197 (2006); Barbieri et al.,Cancer Res 65:2314-2320 (2005); Yuan et al., BMC Cancer 11:57 (2011);Yang et al. Cancer Res 71:3688-3700 (2011)). Examples of useful siRNAsequences for inhibiting Fzd2 are provided herein.

In one embodiment, use of the agent is to thereby result in a decreasein the target Fzd2 mRNA level in a cell by at least about 5%, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 95%, about 99%, about 100% of the mRNA leveltypically measured in the cell in the absence of the RNAi. In oneembodiment, the mRNA levels are decreased by at least about 70%, about80%, about 90%, about 95%, about 99%, or about 100%.

The agent that is a nucleic acid to be expressed in the target cell maycomprise a vector. Many such vectors useful for transferring exogenousgenes into target mammalian cells are available. The vectors may beepisomal, e.g., plasmids, virus derived vectors such cytomegalovirus,adenovirus, etc., or may be integrated into the target cell genome,through homologous recombination or random integration, e.g., retrovirusderived vectors such MMLV, HIV-1, ALV, etc. For modification of stemcells, lentiviral vectors are preferred. Lentiviral vectors such asthose based on HIV or FIV gag sequences can be used to transfectnon-dividing cells, such as the resting phase of human stem cells (seeUchida et al. (1998) P.N.A.S. 95(20): 11939-44). As used herein, theterm “vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. Preferred vectors arethose capable of autonomous replication and/or expression of nucleicacids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”.

Delivery of Agents to the Cell

The agent is contacted to the cell such that it can exert its intendedeffect on the cell. In one embodiment, the agent exerts its effects onthe cells merely by interacting with the exterior of the cell (e.g., bybinding to a receptor, such as with an antibody or antigen bindingfragment). Agents that act on the cell internally (e.g., RNAi) may bedelivered in a form that is readily taken up by the cell when contactedto the cell (e.g., in a formulation which facilitates cellular uptakeand delivery to the appropriate subcellular location). In oneembodiment, the agent is in a formulation in which it is readily takenup by the cell so that it can exert it effect.

Colloidal dispersion systems may be used as delivery vehicles and toenhance the in vivo stability of the agent to a particular organ, tissueor cell type. Colloidal dispersion systems include, but are not limitedto, macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system is aplurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layers made up of lipidsarranged in a bilayer configuration (see, generally, Chonn et al.,Current Op. Biotech. 1995, 6, 698-708). Other examples of cellularuptake or membrane-disruption moieties include polyamines, e.g.spermidine or spermine groups, or polylysines; lipids and lipophilicgroups; polymyxin or polymyxin-derived peptides; octapeptin; membranepore-forming peptides; ionophores; protamine; aminoglycosides; polyenes;and the like. Other potentially useful functional groups includeintercalating agents; radical generators; alkylating agents; detectablelabels; chelators; or the like.

Other colloidal dispersion systems lipid particle or vesicle, such as aliposome or microcrystal, may be suitable for administration. Theparticles may be of any suitable structure, such as unilamellar orplurilamellar, so long as the antisense oligonucleotide is containedtherein. Positively charged lipids such asN—[I-(2,3dioleoyloxi)propyl1-N,N,N-trimethyl-anunoniummethylsulfate, or“DOTAP,” are particularly preferred for such particles and vesicles. Thepreparation of such lipid particles is well known. See, e.g., U.S. Pat.Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and4,921,757, which are incorporated herein by reference. Other non-toxiclipid based vehicle components may likewise be utilized to facilitateuptake of the nucleic acid compound by the cell.

In some embodiments, in order to increase nuclease resistance in an RNAiagent as disclosed herein, one can incorporate non-phosphodiesterbackbone linkages, as for example methylphosphonate, phosphorothioate orphosphorodithioate linkages or mixtures thereof, into one or morenon-RNASE H-activating regions of the RNAi agents. Such non-activatingregions may additionally include 2′-substituents and can also includechirally selected backbone linkages in order to increase bindingaffinity and duplex stability. Other functional groups may also bejoined to the oligonucleoside sequence to instill a variety of desirableproperties, such as to enhance uptake of the oligonucleoside sequencethrough cellular membranes, to enhance stability or to enhance theformation of hybrids with the target nucleic acid, or to promotecross-linking with the target (as with a psoralen photo-cross-linkingsubstituent). See, for example, PCT Publication No. WO 92/02532, whichis incorporated herein in by reference.

Methods of delivering RNAi interfering (RNAi) agents, e.g., an siRNA, orvectors containing an RNA interfering agent, to the target cells (e.g.,horizontal basal cells) can include, for example (i) injection of acomposition containing the RNA interfering agent, e.g., an siRNA, or(ii) directly contacting the cell, with a composition comprising an RNAinterfering agent, e.g., an siRNA. In another embodiment, RNAinterfering agents, e.g., an siRNA can be injected directly into anyblood vessel, such as vein, artery, venule or arteriole, via, e.g.,hydrodynamic injection or catheterization. In some embodiments RNAiagents such as siRNA can delivered locally to specific organs or bysystemic administration.

Pharmaceutical Compositions

In one embodiment, the agent described herein is an active ingredient ina composition comprising a pharmaceutically acceptable carrier (referredto herein as a pharmaceutical composition). Such a composition isreferred to herein as a pharmaceutical composition. A “pharmaceuticallyacceptable carrier” means any pharmaceutically acceptable means to mixand/or deliver the targeted delivery composition to a subject. The term“pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agents fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the composition and is compatible withadministration to a subject, for example a human. Such compositions canbe specifically formulated for administration via one or more of anumber of routes, such as the routes of administration described herein.Supplementary active ingredients also can be incorporated into thecompositions. When an agent, formulation or pharmaceutical compositiondescribed herein, is administered to a subject, preferably, atherapeutically effective amount is administered. As used herein, theterm “therapeutically effective amount” refers to an amount that resultsin an improvement or remediation of the condition.

Administration

Administration of the pharmaceutical composition is by means which theagent contained therein will contact the target cell or tissue (e.g.,tumor or cancer). Examples of such routes are localized and systemic,which include, without limitation parenteral, enteral, and topicaladministration. Parenteral administration is usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, intratumoral, and intrasternal injection andinfusion. Administration can be systemic administration, or localized,as determined necessary by the skilled practitioner. Localizedadministration can be directed to the location of the target tissue.Pharmaceutical compositions and formulations for specified modes ofadministration, described herein are also encompassed by the presentinvention. The compounds of the invention can be administeredparenterally by injection or by gradual infusion over time and can bedelivered by peristaltic means.

The agents described herein are administered to a subject by routes andin formulations that will deliver the agent to tumor tissue or cells ofthe subject. Delivery to the tumor cell refers to eventual contacting ofthe cancer cells by the agent, in a manner required for the agent toexert the intended effect. For instance, an antibody or antigen bindingfragment thereof is administered by a route, and in a formulation, thatpromotes contact with the Fzd2 receptor located on the exterior(extracellular location) of the cancer cells. The amount delivered tothe subject is sufficient to promote an effective amount of the agentbeing delivered to the cancer cells. The route of administration willdepend upon various factors, including the location of the cancer cells,the desired final concentration at the cancer cell. Preferred routes andlevels of administration, as well as appropriate formulations, will bedetermined by the skilled practitioner for each individual subject.

Administration may be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays, for example, or usingsuppositories. For oral administration, the compounds of the inventionare formulated into conventional oral administration forms such ascapsules, tablets and tonics.

For topical administration, the pharmaceutical composition is formulatedinto ointments, salves, gels, or creams, as is generally known in theart.

The therapeutic compositions of this invention are conventionallyadministered intravenously, as by injection of a unit dose, for example.The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual.

Typically the agent is administered to a subject as a pharmaceuticalcomposition. A pharmaceutical composition contains the active agent,described herein, and a pharmaceutically acceptable carrier. Thecomposition may further comprise one or more additional ingredients,such as additional active agents.

The pharmaceutical composition formulation can further compriseingredients for protection of the active agent in the administeredphysiological and cellular environment. For example, the formulation maycontain an ingredient to protect the agent from degradation by digestiveenzymes, or from attack by the immune system of the subject.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

Downmodulatory agents of Fzd2 (e.g., Anti-Fzd2 peptides and/or Abs) ofthe present invention can be administered either as individualtherapeutic agents or in combination with other therapeutic agents. Theycan be administered alone, but are generally administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adaily dosage of active ingredient can be about 0.01 to 100 milligramsper kilogram of body weight. Ordinarily 1.0 to 5, and preferably 1 to 10milligrams per kilogram per day given in divided doses 1 to 6 times aday or in sustained release form is effective to obtain desired results.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about 0.5to 95% by weight based on the total weight of the composition.

As a non-limiting example, treatment of cancer by the methods describedherein in humans or animals can be provided as a daily dosage of apharmaceutical composition comprising anti-Fzd2 peptides, monoclonalchimeric and/or murine antibodies of the present invention at a dosageof 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, onat least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, orany combination thereof, using single or divided doses of every 24, 12,8, 6, 4, or 2 hours, or any combination thereof.

For parenteral administration, anti-Fzd2 antibodies or antigen bindingfragments can be formulated as a solution, suspension, emulsion orlyophilized powder in association with a pharmaceutically acceptableparenteral vehicle. Examples of such vehicles are water, saline,Ringer's solution, dextrose solution, and 5% human serum albumin.Liposomes and nonaqueous vehicles such as fixed oils can also be used.The vehicle or lyophilized powder can contain additives that maintainisotonicity (e.g., sodium chloride, mannitol) and chemical stability(e.g., buffers and preservatives). The formulation is sterilized bycommonly used techniques.

Definitions

Downmodulation refers to reducing one or more functions of the protein(Fzd2) (e.g., ligand binding, signal transduction, etc.). This can beaccomplished by directly inhibiting the production of functional Fzd2itself in the cell (e.g., by reducing gene expression or proteinsynthesis), and/or by reducing Fzd2 function/activity. Fzd2function/activity can be reduced, for example by directly inhibiting theFzd2 protein itself (e.g. by inhibiting ligand binding) or otherwisetargeting that protein for degradation. As such, an agent useful in thepresent invention is one that inhibits Fzd2 gene expression or proteinsynthesis, or inhibits one or more Fzd2 function or activity.Downmodulation may be accomplished by a reduction of the amount of areceptor on the surface of a cell (e.g., by promotion of internalizationof the receptor). In one aspect of the invention the level of Fzd2expressed on the surface of a cell is reduced by contacting the cellwith an agent that downmodulates Fzd2 in the cell. In one embodiment,the agent binds specifically to Fzd2 of the cell and promotesinternalization of the Fzd2 by the cell. Such agents are describedherein. A significant amount of downmodulation is expected to produceuseful results in the methods described herein. In one embodiment,downmodulation results in at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, or at least 50%, or at least 60%, or at least70%, or at least 80%, or at least 90%, or at least 99% reduction in thelevel of Fzd2 protein or mRNA in the cell by the methods describedherein, as compared to that typically observed or expected in theabsence of treatment.

As used herein, the term “treating” and “treatment” and/or “palliating”refers to administering to a subject an effective amount of the agent,so as to inhibit Fzd2 (e.g., promote receptor internalization and/orinhibit ligand binding), such that the subject has an improvement in thecondition, for example, detectable beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, a decrease in the severity ofthe symptoms, including partial or complete alleviation of one or moresymptoms; prevention or delay in the development of one or moreindicators, symptoms, or markers; diminishment of extent of discomfort(i.e., not worsening) incurred by the condition; slowing of progressionof the condition, amelioration or palliation of the condition, and alsocomplete recovery from the condition. A decrease in onset of one or moreindicators, symptoms, markers of the tumor or cancer as described hereinmay be by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or at least 90%, or at least 99% that typically observed orexpected in the absence of treatment. In one embodiment, the onset ofone or more symptoms, indicators or markers is completely prevented. Inone embodiment, one or more of the symptoms of the tumor or cancerexperienced prior to administration are alleviated by at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, or at least 50%, orat least 60%, or at least 70%, or at least 80%, or at least 90%, or atleast 99% that experienced in the absence of treatment.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically significant reduction in asymptom associated with a disease, disorder or injury being treated,when administered to a typical subject with that condition. Atherapeutically significant reduction in a symptom is, e.g. about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, about 100%, about 125%, about 150% or more as comparedto a control or non-treated subject.

The compositions as disclosed herein can be administered inprophylatically or therapeutically effective amounts. A prophylacticallyeffective amount means that amount necessary, at least partly, to delaythe onset of, inhibit the progression of, or halt altogether, the onsetor progression of the particular disease or disorder being treated.

Such amounts for therapy or prophylaxis will depend, of course, on theparticular condition being treated, the severity of the condition andindividual patient parameters including age, physical condition, size,weight and concurrent treatment. These factors are well known to thoseof ordinary skill in the art and can be addressed with no more thanroutine experimentation. It is preferred generally that a maximum dosebe used, that is, the highest safe dose according to sound medicaljudgment. It will be understood by those of ordinary skill in the art,however, that a lower dose or tolerable dose can be administered formedical reasons, psychological reasons or for virtually any otherreasons.

As used herein, an antigen binding fragment of an antibody includes,without limitation, the Fab, scFv, Fv, dAb, and Fd fragments. Exampleantigen binding fragments are (i) the Fab fragment consisting of the VL,VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH andCH1 domains; (iii) the Fv fragment consisting of the VL and VH domainsof a single arm of an antibody, (iv) the dAb fragment (Ward, E. S. etal., Nature 341, 544-546 (1989) which consists of a VH domain; (v)isolated CDR regions; and (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two Fab fragments linked by a disulphide bridge at the hingeregion. Although the two domains of the Fv fragment are coded for byseparate genes, it has proved possible to make a synthetic linker thatenables them to be made as a single protein chain (known as single chainFv (scFv); Bird, R. E. et al., Science 242, 423-426 (1988) Huston, J. S.et al., Proc. Natl. Acad. Sci., USA 85, 5879-5883 (1988)) by recombinantmethods.

The term “subject” includes organisms which are capable of sufferingfrom a disease, disorder or injury, who could otherwise benefit from theadministration of a compound or composition of the invention, such ashuman and non-human animals. The terms subject, individual, and patientare used interchangeably herein. The term “non-human animals” of theinvention includes all vertebrates, including, without limitation,mammals (e.g., rodent (mice, rat, rabbit, guinea pig), primate, canine,equine, bovine, feline, porcine) and non-mammals. Non-human primates arealso possible subjects. Specific subjects include, without limitation,humans, sheep, dog, cow, horses, chickens, mice, rats, hamster, rabbit,amphibians, reptiles, etc. Cells described herein can be in the contextof or otherwise isolated from any such subject described herein.

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount when compared to an appropriate reference level. Inone embodiment, the reduction is at least 10% as compared to a referencelevel, or at least about 20%, or at least about 30%, or at least about40%, or at least about 50%, or at least about 60%, or at least about70%, or at least about 80%, or at least about 90% or up to and includinga reduction of 100% (e.g. absent level or non-detectable level ascompared to a reference level).

The term “tumor cell” is used to refer to precancerous and alsocancerous cells.

As used herein, the term “administer” refers to the placement of apharmaceutically acceptable composition into a subject by a method orroute which results in at least partial localization of an effectiveamount of the composition to a desired site (e.g., to the tumor tissueor cells of the tumor) such that desired effect is produced. A compoundor composition described herein can be administered by any appropriateroute known in the art including, but not limited to, oral or parenteralroutes, including intravenous, intramuscular, subcutaneous, transdermal,airway (aerosol), pulmonary, nasal, rectal, and topical (includingbuccal and sublingual) administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebrospinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±1%.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the invention, yet open to the inclusion of unspecified elements,essential or not (“comprising). In some embodiments, other elements tobe included in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A method of treating cancer in a subject comprising administering    to the subject a therapeutically effective amount of an antibody or    antigen binding fragment thereof that downmodulates Fzd2, such that    the antibody or antigen binding fragment thereof is delivered to    cancer cells of the subject, to thereby treat the cancer.-   2. A method of inhibiting growth, migration and/or invasion of a    cancer cell in a subject comprising administering to the subject a    therapeutically effective amount of an antibody or antigen binding    fragment thereof that downmodulates Fzd2, such that the antibody or    antigen binding fragment thereof is delivered to the cancer cells,    to thereby treat the cancer.-   3. The method of any one of paragraphs 1 or 2, wherein the antibody    specifically binds Fzd2.-   4. The method of paragraph 3, wherein the antibody binds to Fzd2 and    promotes internalization of the Fzd2 receptor by the cancer cells.-   5. The method of any one of paragraph 3-4, wherein the antibody to    Fzd2 prevents ligand binding to Fzd2.-   6. The method of any one of paragraph 3-5, wherein the antibody    specifically binds an extracellular portion of the Fzd-2 protein.-   7. The method of any one of paragraphs 3-6, wherein the antibody    specifically binds to Fzd2 within a region of Fzd2 corresponding to    amino acids 24-247 of Fzd2.-   8. The method of any one of paragraphs 3-7, wherein the antibody    specifically binds to Fzd2 within a region of Fzd2 corresponding to    amino acids 125-163 of Fzd2.-   9. The method of any one of paragraphs 3-7, wherein the antibody    specifically binds to Fzd2 within a region of Fzd2 corresponding to    amino acids 134-163 of Fzd2.-   10. The method of any one of paragraphs 3-7, wherein the antibody    specifically binds to Fzd2 within a region of Fzd2 corresponding to    amino acids 144-163 of Fzd2.-   11. The method of any one of paragraphs 3-10, wherein the antibody    specifically binds to the epitope HGAEQICVGQNHSEDGAPAL (SEQ ID NO:    1).-   12. The method of any one of paragraphs 3-11, wherein the antibody    is monoclonal.-   13. The method of any one of paragraphs 3-11, wherein the antibody    is polyclonal.-   14. The method of any one of paragraphs 3-12, wherein the antibody    is humanized.-   15. The method of any one of paragraphs 1-14, wherein the cancer is    selected from the group consisting of gastrointestinal cancer,    prostate cancer, ovarian cancer, breast cancer, head and neck    cancer, lung cancer, non-small cell lung cancer, cancer of the    nervous system, kidney cancer, retina cancer, skin cancer, liver    cancer, pancreatic cancer, genital-urinary cancer and bladder    cancer.-   16. The method of any one of paragraphs 1-15, wherein the cancer is    liver cancer.-   17. The method of any one of paragraphs 1-16, wherein the cancer is    late stage hepatocellular carcinoma.-   18. The method of any one of paragraphs 1-17, wherein the cancer    displays overexpression of Fzd2.-   19. The method of any one of paragraphs 1-18, wherein the cancer    displays overexpression of Wnt5a.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1: Frizzled 2 Receptor as a Target for TherapeuticAntibodies in the Treatment of Hepatocellular Carcinoma

Fzd2 is Overexpressed in Late Stage and Poorly Differentiated HCC

Initial studies of Wnt receptor expression in hepatocellular carcinomasrevealed marked overexpression of Frizzled 2 (Fzd2) in late-stage andpoorly differentiated HCC (FIG. 1). The transcript levels of the Fzd2gene were measured in 48 tissue samples obtained from patients withhistopathologically confirmed HCC (stage I, n=7; stage II, n=8; stageIII, n=8; stage IV, n=3; tumor lesion, n=14), as well as in normal liversamples (n=8). Fzd2 was found to be significantly overexpressed inlate-stage HCC (Stage III and IV) compared with normal tissue (P<0.05).Further, the levels of Fzd2 expression correlated negatively with thedegree of tissue differentiation. Moderately and poorly differentiatedtumors showed higher levels of Fzd2 compared with well-differentiatedtumor types. Further, we found that Fzd2 is highly overexpressed inmesenchymal-like, metastatic HCC cell line (FOCUS) compared with themore epithelial-like HepG2 HCC cell line (FIG. 2).

Fzd2 Knockdown or Treatment with an Anti-Fzd2 Antibody Reduces CellularMigration and Invasion In Vitro.

The Wnt proteins are growth factors that have been shown to playcritical roles in proliferation, migration and invasion. The Wntproteins mediate their effects through binding to and activatingreceptors of the Fzd family. siRNA-mediated knockdown of Fzd2 ortreatment with anti-Fzd2 antibody was found to inhibit Wnt-mediatedcellular migration and invasion of FOCUS cells in vitro, suggesting thatFzd2-mediated non-canonical Wnt signaling is critical for theseprocesses (FIG. 3).

The Anti-Fzd2 Antibody Causes Internalization of Cell Surface Fzd2Receptors

Using a cell surface biotinylation assay, it was shown that treatment ofFOCUS cells with the anti-Fzd2 antibody causes internalization anddegradation of Fzd2. Specifically, Fzd2 was found to be expressed at thecell membrane in the absence of its cognate ligand, Wnt5a. The proteinlevels of Fzd2 at the cell surface markedly decreased upon Wnt5astimulation or treatment with the anti-Fzd2 antibody (FIG. 4).

Fzd2 Knockdown Reduces Tumor Growth in Nude Mice

To understand the extent of Fzd2 alterations in the pathogenesis of HCC,FOCUS were cells subcutaneously injected into athymic mice and theability of cells to form tumor outgrowths were monitored. FOCUS cellsgrew rapidly in this model, producing measurable tumors by day 4. Whenthe outgrowths were approximately 200 mm³, mice were divided at randominto two groups (control and treated). The treated group receivedFzd2-siRNA injection on alternate days (MWF) for two weeks, while thecontrol group received s.c injection of in-vivo transfection reagentonly. In the presence of siRNA directed against Fzd2, tumor growth wassignificantly slower with a notable lag in the exponential growth phase(FIG. 5). Interestingly, the tumors in the treated group started to growrapidly when the Fzd2-siRNA injections were discontinued. These datasuggest that the highly tumorigenic potential of FOCUS cells is, inpart, mediated through Fzd2.

Overall, the in vitro and in vivo data suggest that Fzd2 is an oncogenein HCC and that overexpression of Fzd2 contributes to the progressionand metastasis of HCC.

Epitope Mapping of Anti-Fzd2 Antibody

To map the epitope of anti-Fzd2 antibody (R & D Systems; MAB1307), threeoverlapping constructs spanning from residues 26-172 in the N-terminusregion of Fzd2 were designed (FIG. 6A). When overexpressed in HEK293cells, only the fragment 1 (full length, aa 26-172) and fragment 3(C-terminus region, aa 100-172) were recognized by anti-Fzd2 antibodysuggesting that the epitope of anti-Fzd2 antibody lies within theFragment 3 region (FIG. 6A). To further map the epitope of anti-Fzd2antibody, three overlapping peptides spanning Fragment 3 weresynthesized. Using the peptide array, only Peptide 2 (aa 134-163) wasseen to be recognized by the anti-Fzd2 antibody (FIG. 6 B). Since,anti-Fzd2 antibody did not recognize Peptide 1 (aa 100-132) oroverlapping Peptide 2 (aa 125-143), the epitope for anti-Fzd2 antibodywas determined to lie within the residues134-ERLRCEHFPRHGAEQICVGQNHSEDGAPAL-163 (SEQ ID NO: 1) in the N-terminusregion of Fzd2. This epitope lies in the region which is highly specificto Fzd2 and does not share homology with other Fzd family memberssuggesting high specificity of anti-Fzd2 antibody. These sequencescorrespond to that shown for human fzd-2 in NCBI reference NP_001457.

Materials and Methods

Cell Lines and Reagents

Liver cancer cell line HepG2 and HEK 293T cells were obtained fromAmerican Type Culture Collection (ATCC, Rockville, Md.). FOCUS cellswere obtained from J. Wands (Brown University) and have been describedpreviously [5]. All cell lines were maintained in Dulbecco's ModifiedEagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum(FBS), 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin.

Signal Silence Fzd2 siRNA was purchased from Santa Cruz Biotechnology(Santa Cruz, Calif.). siRNA was introduced into cells usingLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. Recombinant human Wnt5a was from R&D Systems (Minneapolis,Minn.). Primary antibodies were obtained from the following sources:rabbit anti-β-catenin (Cell Signaling Technology), goat anti-Fzd2 (SantaCruz Biotechnology), rat anti-Fzd2 (R&D Systems), rabbit anti-Fzd2(Abcam).

RNA Extraction and Quantitative Real-Time PCR

HepG2 and FOCUS cells were serum-starved for 24 h and total cellular RNAwas isolated using an RNeasy Mini Kit (QIAGEN, Santa Clara, Calif.).mRNA levels for the 84 Wnt-related genes were determined using the RT2Profiler™ qPCR array (SA Biosciences Corporation, Frederick, Md.).Briefly, 1 μg of total RNA was reverse transcribed into first strandcDNA using an RT2 First Strand Kit (SA Biosciences). The resulting cDNAwas subjected to qPCR using human gene-specific primers for 96 differentgenes, including the 84 Wnt-related genes and five housekeeping genes(B2M, HPRT1, RPL13A, GAPDH, and ACTB). The qPCR reaction was performedwith an initial denaturation step of 10 min at 95° C., followed by 15 sat 95° C. and 60 s at 60° C. for 40 cycles using an Mx3000P™ QPCR system(Stratagene, La Jolla, Calif.).

The mRNA levels of each gene were normalized relative to the mean levelsof the five housekeeping genes and compared with the data obtained fromunstimulated, serum-starved cells using the 2−ΔΔCt method. According tothis method, the normalized level of a mRNA, X, is determined usingequation 1: (1)X=2^(−Ct(GOI))/2^(−Ct(CTL))  (1)

where Ct is the threshold cycle (the number of the cycle at which anincrease in reporter fluorescence above a baseline signal is detected),GOI refers to the gene of interest, and CTL refers to a controlhousekeeping gene. This method assumes that Ct is inversely proportionalto the initial concentration of mRNA and that the amount of productdoubles with every cycle.

TissueScan Oncology Panel Arrays

A collection of 48 cDNA samples derived from tumor biopsies was obtainedfrom OriGene Technologies Inc. (Rockville, Md.). The samples representedall four stages of liver carcinoma, as well as normal tissue. Geneexpression was assessed by qPCR as described above. The cancer data werenormalized relative to the data collected from the normal tissue samplesand analyzed using the Kruskal-Wallis test at a significance level of0.05.

Cell Migration Assay

Cell migration was assessed using a QCM™ chemotaxis 96-well cellmigration assay kit (Chemicon, Temecula, Calif.). Briefly, FOCUS cellstransiently transfected with Fzd2-siRNA or Control siRNA for 48 hourswere suspended in DMEM and plated in the top chamber. DMEM containingWnt5a (200 ng/mL) was added to the bottom chamber for 2 hours. Migratorycells in the bottom chamber were dissociated from the membrane, lysed,and quantified by adding CyQuant GR dye. Measurements were performed intriplicate and normalized to control cells.

Similar cell migration assays were performed utilizing the anti-Fzd2antibody in place of transient transfection with the Fzd2-siRNA toinhibit Fzd2. The anti-Fzd2 antibody was added to the cell culturemedium to a final concentration of 10 μg/ml for 2 hours prior to themigration assays.

Cell Invasion Assay

Cell invasion assay was measured using a CytoSelect™ Cell Invasion Assay(Cell Biolabs) according to the manufacturer's instructions. Briefly,FOCUS cells transiently transfected with Fzd2-siRNA or Control siRNA for48 hours were seeded and allowed to invade towards Wnt5a (200 ng/mL) fortwo hours. Invasive cells, on the bottom of the invasion membrane, werestained and then quantified by adding CyQuant GR dye. Measurements wereperformed in triplicate and normalized to control cells.

Similar cell invasion assays were performed utilizing the anti-Fzd2antibody in place of transient transfection with the Fzd2-siRNA toinhibit Fzd2. The anti-Fzd2 antibody was added to the cell culturemedium to a final concentration of 10 μg/ml for 2 hours prior to themigration assays.

Surface Biotynlation Assay

Biotinylation of surface proteins was performed as described [6].Briefly, FOCUS cells treated with either PBS (Control) Wnt5a (100 ng/ml)or Anti-Fzd2 antibody (10 μg/ml final concentration) for 1 hour werewashed twice with PBS containing 1 mM MgCl2 and 0.1 mM CaCl2 andincubated in biotinylation buffer (154 mM NaCl, 10 mM Hepes [pH 7.6], 3mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM glucose, 0.5 mg/ml EZ Link SulfoHNS-SS-Biotin (Thermo Scientific, Rockford, Ill.) for 40 minutes at 4°C. Cells were then incubated in PBS containing 100 mM glycine for 5minutes at 4° C., followed by one wash in PBS containing 1 mM MgCl2 and0.1 mM CaCl2. Cells were lysed, and total protein concentration wasdetermined using the BCA protein assay (Pierce, Rockford, Ill.).Biotinylated proteins were immunoprecipitated with streptavidin beadsand total surface Fzd2 determined by SDS-PAGE and western blotting, asdescribed below.

Tumorigenicity in Nude Mice

All in vivo experiments were performed using 6-week-old to 8-week-oldathymic nude mice (NIH. Mice were maintained in laminar flow rooms withconstant temperature and humidity. FOCUS cells were inoculated s.c. intoeach flank of the mice. Cells (2×10⁶ in suspension) were injected on day0, and tumor growth was followed every 2 to 3 days by tumor diametermeasurements using vernier calipers. Tumor volumes (V) were calculatedusing the formula: V=AB2/2 (A, axial diameter; B, rotational diameter).When the outgrowths were approximately 200 mm³, mice were divided atrandom into two groups (control and treated). The treated group receivedFzd2-siRNA injection on alternate days (MWF) for two weeks, while thecontrol group received s.c injection of in-vivo transfection reagentonly. In the presence of siRNA directed against Fzd2, tumor growth wassignificantly slower with a notable lag in the exponential growth phase.

Protein Isolation and Quantitative Western Blotting

Cells were rinsed in Phosphate Buffered Saline (PBS) and lysed in LysisBuffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100 (v/v), 2 mM EDTA,pH 7.8 supplemented with 1 mM sodium orthovanadate, 1 mMphenylmethylsulfonyl fluoride (PMSF), 10 μg/mL aprotinin, and 10 μg/mLleupeptin). Protein concentrations were determined using the BCA proteinassay (Pierce, Rockford, Ill.) and immunoblotting experiments wereperformed using standard procedures. For quantitative immunoblots,primary antibodies were detected with IRDye 680-labeled goat-anti-rabbitIgG or IRDye 800-labeled goat-anti-mouse IgG (LI-COR Biosciences,Lincoln, Nebr.) at 1:5000 dilution. Bands were visualized and quantifiedusing an Odyssey Infrared Imaging System (LI-COR Biosciences).

Epitope Mapping of Anti-Fzd Antibody

To map the epitope of anti-Fzd2 antibody, three overlapping fragmentsspanning from residues 26-172 were constructed in pcDNA3.1 mammalianexpression vector. These constructs were transiently transfected inHEK293 cells and whole cell lysates collected after 48 hours weresubjected to western blotting with anti-Fzd2 antibody as describedabove. To further map the region of Fragment 3 (aa 100-172), threeoverlapping peptides were synthesized commercially (LifeTein, NJ). Thesepeptides and lysates from HEK293 cells expressing Fzd2 full length geneor empty vector (negative) were printed on nitrocellulose coated glassslides using Aushon 2470 arrayer. The peptide array slide was blockedwith 3% (v/v) BSA and probed with anti-Fzd2 antibody. Spots werevisualized and quantified using an Odyssey Infrared Imaging System(LI-COR Biosciences).

REFERENCES

-   1. Parkin, D., F. Bray, and S. Devesa, Cancer burden in the    year 2000. The global picture. European Journal of Cancer, 2001.    37: p. 4-66.-   2. Carr, B., Hepatocellular carcinoma: current management and future    trends. Gastroenterology, 2004. 127(5): p. S218-S224.-   3. Lee, H., M. Kim, and J. Wands, Wnt/Frizzled signaling in    hepatocellular carcinoma. Front Biosci, 2006. 11: p. 1901-1915.-   4. Reya, T. and H. Clevers, Wnt signalling in stem cells and cancer.    Nature, 2005. 434(7035): p. 843-50.-   5. He, L., et al., Establishment and characterization of a new human    hepatocellular carcinoma cell line. In Vitro, 1984. 20(6): p.    493-504.-   6. Smith, C. A., et al., The cell fate determinant numb interacts    with EHD/Rme-1 family proteins and has a role in endocytic    recycling. Mol Biol Cell, 2004. 15(8): p. 3698-708.

Example 2: Wnt Receptor: A Novel Biologics Target Against Liver Cancer

Fzd2 Knockdown or Treatment with an Anti-Fzd2 Antibody Reduces CellularMigration and Invasion In Vitro.

Knockdown of Fzd2 by RNAi or exposure of cells to an anti-Fzd2 antibodywas shown to reduce cellular motility and invasiveness of HCC cells andreverts oncogenic phenotypes to a normal epithelial-like state (FIG. 8).Using a cell surface biotinylation assay, treatment of FOCUS HCC cellswith an anti-Fzd2 antibody was found to cause internalization anddegradation of Fzd2 (FIG. 8). Upon Wnt5a stimulation, cell surfacelevels of Fzd2 decreased markedly, and this down-regulation is alsoobserved in cells treated with an anti-Fzd2 antibody. The antibody,however, does not activate downstream signaling.

Fzd2 Knockdown Induces Cytostasis in a Xenograft Model of HCC in Mice

To understand the extent of Fzd2 alterations in the pathogenesis of HCC,FOCUS cells were subcutaneously injected into athymic mice and theability of the cells to form tumor outgrowths was monitored. FOCUS cellsgrew rapidly in this xenograft tumor model, producing measurable tumorsby day 4. When the tumors were ˜200 mm³, mice were divided at randominto two groups (control and treated). The treated group receivedFzd2-siRNA injections on alternate days (MWF) for two weeks, whereas thecontrol group received s.c injections of in vivo transfection reagentonly. In the presence of siRNA directed against Fzd2, tumor growth wassignificantly slower with a notable lag in the exponential growth phase(FIG. 8E). Tumors in the treated group resumed rapid growth after theFzd2-siRNA injections were discontinued. These data suggest that thehighly tumorigenic potential of FOCUS cells is, in part, mediated byFzd2 and that Fzd2-siRNA induces cytostasis rather than cell death.Similarly, stable knockdown of Fzd2 using shRNA against Fzd2 alsoreduced the tumor growth in nude mice (FIG. 8F).

Overall, the in vitro and in vivo data suggest that Fzd2 is an oncogenein HCC and that overexpression of Fzd2 contributes to the progression ofHCC. The fact that Fzd2 causes cytostasis rather than tumor cell deathis typical of targeted drugs that bind growth factor receptors. In thecase of anti-ErbB drugs, for example, it has been shown that cytostasiscan result in effective tumor inhibition in human patients.

Kaplan-Meier Survival Analysis for 40 HCC Patients

Kaplan-Meier survival data indicate that metastatic HCC patients hadhigh expression of Fzd2 receptor which correlated with substantiallyshorter survival (p=0.0053) than metastasis-free patients who had lowexpression of Fzd2 (FIG. 9).

Binding Analysis of Fzd2 Specific Antibody

Binding analysis was performed for the epitope mapped frizzled-2antibody (discussed in Example 1) and frizzled-2. The results of theanalysis are shown below in Table 1 and FIG. 10. The antibody wasdetermined to have high affinity binding to Fzd2 (<20 nM).

The siRNA and shRNA studies together with the Kaplan-Meier survivalanalysis derived from the clinical samples and outcomes of HCC patientshave demonstrated that Fzd2 is an excellent target in HCC. In addition,we have shown that binding to the epitope identified here by acommercial antibody causes receptor internalization and inactivation ofthe downstream signaling pathway (e.g. ↓ pStat3).

TABLE 1 ka Rmax Analyte KA Ligand Analyte (1/Ms) kd (1/s) (RU)Concentration (1/M) KD (M) Chi2 Frizzled-2 Ab Frizzled-2 3.9 × 10⁴ 7.4 ×10⁻⁴ 43.1 0-500 nM 5.2 × 10⁷ 1.9 × 10⁻⁸ 0.45Materials and Methods:Cell Lines and Reagents

Liver cancer cell lines SNU449, SNU475, and HepG2 cells were obtainedfrom American Type Culture Collection (ATCC, Rockville, Md.). FOCUScells were obtained from J. Wands (Brown University) and have beendescribed previously [1]. All cell lines were maintained in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovineserum (FBS), 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mLstreptomycin.

Signal Silence Fzd2 siRNA was purchased from Santa Cruz Biotechnology(Santa Cruz, Calif.). siRNA was introduced into cells usingLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. Recombinant human Wnt5a was from R&D Systems (Minneapolis,Minn.). Primary antibodies were obtained from the following sources:rabbit anti-β-catenin (Cell Signaling Technology), goat anti-Fzd2 (SantaCruz Biotechnology), rat anti-Fzd2 (R&D Systems), rabbit anti-Fzd2(Abcam).

Generation of Fzd2 Knockdown Stable Cell Lines

Cell lines were transfected with Fzd2-shRNA constructs (Open Biosystems)and 48 hour post-transfection selected in 4 μg/ml puramycin(Invitrogen). The clones were sorted by FACS and screened for Fzd2knockdown by Western blot. Stable cell lines were maintained in DMEMsupplemented with 10% FBS, and 2 μg/ml puramycin.

RNA Extraction and Quantitative Real-Time PCR

HepG2 and FOCUS cells were serum-starved for 24 h and total cellular RNAwas isolated using an RNeasy Mini Kit (QIAGEN, Santa Clara, Calif.).mRNA levels for the 84 Wnt-related genes were determined using the RT2Profiler™ qPCR array (SA Biosciences Corporation, Frederick, Md.).Briefly, 1 μg of total RNA was reverse transcribed into first strandcDNA using an RT2 First Strand Kit (SA Biosciences). The resulting cDNAwas subjected to qPCR using human gene-specific primers for 96 differentgenes, including the 84 Wnt-related genes and five housekeeping genes(B2M, HPRT1, RPL13A, GAPDH, and ACTB). The qPCR reaction was performedwith an initial denaturation step of 10 min at 95° C., followed by 15 sat 95° C. and 60 s at 60° C. for 40 cycles using an Mx3000P™ QPCR system(Stratagene, La Jolla, Calif.).

The mRNA levels of each gene were normalized relative to the mean levelsof the five housekeeping genes and compared with the data obtained fromunstimulated, serum-starved cells using the 2−ΔΔCt method. According tothis method, the normalized level of a mRNA, X, is determined usingequation 1: (1)X=2^(−Ct(GOI))/2^(−Ct(CTL))  (1)

where Ct is the threshold cycle (the number of the cycle at which anincrease in reporter fluorescence above a baseline signal is detected),GOI refers to the gene of interest, and CTL refers to a controlhousekeeping gene. This method assumes that Ct is inversely proportionalto the initial concentration of mRNA and that the amount of productdoubles with every cycle.

Cell Migration Assay

Cell migration was assessed using a QCM™ chemotaxis 96-well cellmigration assay kit (Chemicon, Temecula, Calif.). Briefly, FOCUS cellstransiently transfected with Fzd2-siRNA or Control siRNA for 48 hourswere suspended in DMEM and plated in the top chamber. DMEM containingWnt5a (200 ng/mL) was added to the bottom chamber for 2 hours. Migratorycells in the bottom chamber were dissociated from the membrane, lysed,and quantified by adding CyQuant GR dye. Measurements were performed intriplicate and normalized to control cells.

Kinetic Wound-Healing Assay

The effect of Fzd2 knockdown on migration of FOCUS cells was studiedusing a wound-healing assay. FOCUS cells were plated on 96-well plates(Essen ImageLock, Essen Instruments, MI, US) and a wound was scratchedwith wound scratcher (Essen Instruments). Small molecule inhibitors atdifferent doses were added immediately after wound scratching and woundconfluence was monitored with Incucyte Live-Cell Imaging System andsoftware (Essen Instruments). Wound closure was observed every hour for48-72 ␣h by comparing the mean relative wound density of at least fourbiological replicates in each experiment.

Cell Invasion Assay

Cell invasion assay was measured using a CytoSelect™ Cell Invasion Assay(Cell Biolabs) according to the manufacturer's instructions. Briefly,FOCUS cells transiently transfected with Fzd2-siRNA or Control siRNA for48 hours were seeded and allowed to invade towards Wnt5a (200 ng/mL) fortwo hours. Invasive cells, on the bottom of the invasion membrane, werestained and then quantified by adding CyQuant GR dye. Measurements wereperformed in triplicate and normalized to control cells.

Surface Biotynlation Assay

Biotinylation of surface proteins was performed as described [2].Briefly, FOCUS cells treated with either PBS (Control) Wnt5a (100 ng/ml)or Anti-Fzd2 antibody for 1 hour were washed twice with PBS containing 1mM MgCl2 and 0.1 mM CaCl2 and incubated in biotinylation buffer (154 mMNaCl, 10 mM Hepes [pH 7.6], 3 mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 10 mMglucose, 0.5 mg/ml EZ Link Sulfo HNS-SS-Biotin (Thermo Scientific,Rockford, Ill.) for 40 minutes at 4° C. Cells were then incubated in PBScontaining 100 mM glycine for 5 minutes at 4° C., followed by one washin PBS containing 1 mM MgCl2 and 0.1 mM CaCl2. Cells were lysed, andtotal protein concentration was determined using the BCA protein assay(Pierce, Rockford, Ill.). Biotinylated proteins were immunoprecipitatedwith streptavidin beads and total surface Fzd2 determined by SDS-PAGEand western blotting, as described below.

Tumorigenicity in Nude Mice

All in vivo experiments were performed using 6-week-old to 8-week-oldathymic nude mice (NIH. Mice were maintained in laminar flow rooms withconstant temperature and humidity. FOCUS cells were inoculated s.c. intoeach flank of the mice. Cells (2×10⁶ in suspension) were injected on day0, and tumor growth was followed every 2 to 3 days by tumor diametermeasurements using vernier calipers. Tumor volumes (V) were calculatedusing the formula: V=AB2/2 (A, axial diameter; B, rotational diameter).When the outgrowths were approximately 200 mm³, mice were divided atrandom into two groups (control and treated). The treated group receivedFzd2-siRNA injection on alternate days (MWF) for two weeks, while thecontrol group received s.c injection of in-vivo transfection reagentonly. In the presence of siRNA directed against Fzd2, tumor growth wassignificantly slower with a notable lag in the exponential growth phase.

Protein Isolation and Quantitative Western Blotting

Cells were rinsed in Phosphate Buffered Saline (PBS) and lysed in LysisBuffer (20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100 (v/v), 2 mM EDTA,pH 7.8 supplemented with 1 mM sodium orthovanadate, 1 mMphenylmethylsulfonyl fluoride (PMSF), 10 μg/mL aprotinin, and 10 μg/mLleupeptin). Protein concentrations were determined using the BCA proteinassay (Pierce, Rockford, Ill.) and immunoblotting experiments wereperformed using standard procedures. For quantitative immunoblots,primary antibodies were detected with IRDye 680-labeled goat-anti-rabbitIgG or IRDye 800-labeled goat-anti-mouse IgG (LI-COR Biosciences,Lincoln, Nebr.) at 1:5000 dilution. Bands were visualized and quantifiedusing an Odyssey Infrared Imaging System (LI-COR Biosciences).

Epitope Mapping of Anti-Fzd Antibody

To map the epitope of anti-Fzd2 antibody, three overlapping fragmentsspanning from residues 26-172 were constructed in pcDNA3.1 mammalianexpression vector. These constructs were transiently transfected inHEK293 cells and whole cell lysates collected after 48 hours weresubjected to western blotting with anti-Fzd2 antibody as describedabove. To further map the region of Fragment 3 (aa 100-172), threeoverlapping peptides were synthesized commercially (LifeTein, NJ). Thesepeptides and lysates from HEK293 cells expressing Fzd2 full length geneor empty vector (negative) were printed on nitrocellulose coated glassslides using Aushon 2470 arrayer. The peptide array slide was blockedwith 3% (v/v) BSA and probed with anti-Fzd2 antibody. Spots werevisualized and quantified using an Odyssey Infrared Imaging System(LI-COR Biosciences).

Kaplan-Meier Survival Analysis

Kaplan Meier survival curves of 40 HCC patients were generated usingpreviously published dataset [3].

Cignal Finder 45-Pathway Reporter Array

Cignal™ 45-Pathway Reporter Arrays (SABiosciences, Frederick, Md.) wereused to simultaneously assess 45 different signaling pathways in FOCUScells expressing GFP or Fzd2-shRNA according to manufacturer'sinstructions. Briefly, reporter DNA constructs in each plate well ofCignal Finder 96-well plates were resuspended with 50 μl Opti-MEM andthen mixed with 50 μl diluted Lipofectamine transfection reagent. Cellswere suspended in Opti-MEM and seeded into wells (10,000 cells/well) forintroducing pathway reporters into cells via reverse transfection. Thecells were incubated for 48 h at 5% CO2 and 37° C. Cells were then lysedand firefly and Renilla (internal transfection control) luciferaseactivity were measured by quantitative luminescence assays (Dual Glo,Promega).

Generation of STA T3 Reporter Cell Line

A Cignal Lenti STAT3 Reporter kit (SABiosciences) was used to transducelenti reporters into cells for evaluation of STAT3 pathway activationaccording to the manufacturer's protocols. Briefly, FOCUS cells wereseeded into wells (200,000 cells/well) in 6-well plates and were grownat 37° C. for 24 h in a humidified 5% CO₂ incubator. Then, the cellswere transduced with Lenti CMV Reporter or Lenti STAT3 Reporter (100μl/well of lentiviral particles) and 2 ml growth medium withoutantibiotics containing 10 μg/ml SureEntry transduction reagent for 24 hand then incubated in fresh growth medium for another 24 h at 37° C. and5% CO₂. Thereafter, the cells were treated with 4.0 μg/ml puromycin ingrowth medium for selection of transduced cells. The puromycin-resistantcell colonies were expanded in 100 mm cell culture dishes and maintainedin DMEM supplemented with 10% FBS, and 2 μg/ml puramycin.

Cytokine Array

FOCUS cells expressing GFP-shRNA or Fzd2-shRNA were cultured inserum-free DMEM for 48 hours, and then cytokine levels in culturesupernatant were detected using the Human Cytokine Array Kit (R&DSystems, Minneapolis, Minn.) per the manufacturer's protocol. Briefly,supernatants were incubated at 4 C over night with membranes arrayedwith antibodies against 36 cytokines. After washing twice, membraneswere incubated for 2 hours with biotin-conjugated primary anti-cytokineantibodies and then washed twice. Membranes were then incubated withIRDye 800CW Streptavidin (LI-COR) for 1 hour at room temperature, andwashed twice. Signals were detected and quantified using an OdysseyInfrared Imaging System (LI-COR Biosciences).

REFERENCES FOR EXAMPLE 2

-   1. He, L., et al., Establishment and characterization of a new human    hepatocellular carcinoma cell line. In Vitro, 1984. 20(6): p.    493-504.-   2. Smith, C. A., et al., The cell fate determinant numb interacts    with EHD/Rme-1 family proteins and has a role in endocytic    recycling. Mol Biol Cell, 2004. 15(8): p. 3698-708.-   3. Ye, Q. H., et al., Predicting hepatitis B virus-positive    metastatic hepatocellular carcinomas using gene expression profiling    and supervised machine learning. Nature medicine, 2003. 9(4): p.    416-23.

Example 3: Pharmacodynamic Biomarkers for Liver Cancer Therapeutics

It has been determined that Fzd2-mediated migration and invasion is, atleast partially, due to the release of several proteases from the serineprotease of the plasminogen/plasminogen activator system (PAI-1) and thefamily of matrix metalloproteinases (MMP2, MMP3 and MMP9). Theexpression and release of these proteases highly correlates with theexpression of Fzd2 receptors in late-stage HCC cell lines (p<0.01). Inaddition, a novel signaling pathway downstream of Fzd2 which includesStat3 and src family kinases, has been elucidated. Specifically,decreases in phosphorylation status of src family kinases and Stat3 aswell as its transcription activity are associated with Fzd-2 knockdownby small interferences or antibody treatment. Thus, phosphorylationstatus of stat3 and src family kinases can be used as proximalpharmacodynamic biomarkers for Fzd2 therapeutics.

MMPs

MMPs, particularly MMPs9, 2 and 3 have been implicated in cancer formore than 40 years. In addition to their role in ECM degradation,mounting evidence suggest their role in angiogenesis, lymphangiogenesisand vasculogenesis which are critical to cancer cell invasion andmetastasis. For example, MMP9 increases the bioavailability ofsequestered VEGF binding to its receptor in several cancers such ascolon and pancreatic cancers. MMP9 also mediates the proteolyticactivation of TGF-β which is an important grow factor in HCC.

PAI-1 (SerpinE1)

Plasminogen is another important system in ECM degradation. However,surprisingly high, rather than low, levels of plasminogen activatorinhibitor (PAI-1) are predictive of poor survival prognosis for patientssuffering from a variety of different cancers. This apparent paradoxicalrole of PAI-1 has demonstrated to promote tumor growth and angiogenesisboth in vitro and in vivo. It has been shown that tight control ofproteolytic breakdown by PAI-1 is essential for the formation of newblood vessels. In addition, by blocking the interaction betweenvitronectin, uPAR, and integrins, PAI-1 may induce cell detachment fromthe extracellular matrix and thereby promote cellular migration andtumor invasion.

sICAM-1

Expression of intracellular adhesion molecule-1 (ICAM-1) has beenestablished to correlate with poor prognosis of solid tumors. A causalrole of ICAM-1 in invasion of metastatic cancers has been shown incancers such as breast and lung. ICAM-1 expression at the cell surfacedictates a tumor's metastatic potential via recruiting and activating aseries of macrophages and neutrophils which lead to the break down ofendovascular and endolymphatic barriers and permit transendothelialtumor cell migration. Soluble form of ICAM-1 (sICAM-1) present in thecirculation is a direct marker for ICAM-1 which is difficult to assessclinically.

STAT3

Deregulated expression of Stat3, a signaling molecule and atranscription factor, has been implicated in ˜80% of cancer, leading tocancer cell proliferation and invasion.

Src Family Kinases

Deregulation of Src family kinases, which can phosphorylate Stat3, havebeen implicated in many types of cancer. The discovery of these secretedproteins and signaling molecules downstream of Fzd2 not only adds to themechanistic details of how Fzd2 promotes tumor cell migration andinvasion, but can be used as pharmacodynamic markers for therapeuticswhich target Fzd2 pathway

Fzd2 Mediates Release of SerpinE1 and sICAM1

Human cytokine arrays were generated and used to analyze proteinexpression. The arrays consisted of antibodies spotted in duplicate ontoa nitrocellulose membrane to allow high-throughput multi-analyteprofiling of 36 cytokines, chemokines, and acute phase proteins incondition media. Conditioned media from FOCUS-WT or FOCUS-shFzd2 cellswas mixed with a cocktail of biotinylated detection antibodies, and thenincubated with the Human Cytokine Array. The array was then incubatedwith Infrared-800 labeled-streptavidin followed by infra-red detectionusing LiCor odysseys imaging system. Results of the analysis are shownin FIG. 11. Images of human cytokine array probed with conditioned mediafrom FOCUS-WT (top of FIG. 11A) and FOCUS-shFzd2 (bottom of FIG. 11A).After detection, the array data were quantified to generate a proteinprofile. The abundance of 36 cytokines measured using this assay isshown in FIG. 11B. The amount of SerpinE1 and siCAM1 released wassignificantly lower in the condition media from FOCUS-shFzd2 cells.

Matrix Metalloproteinases (MMPs) and SerpinE1 are Overexpressed in LateStage, Aggressive and Poorly Differentiated Hepatocellularcarcinoma(HCC) Lines

Analysis was performed to examine the relative mRNA expression ofvarious MMPs and SerpinE1 in early stage nonagressive (HepG2 and Huh7)and late stage, aggressive (SNU475 and FOCUS) cell lines. Results areshown in FIG. 12.

Fzd2 Regulates the mRNA Expression of Matrix Metalloproteinases (MMPs)and SerpinE1

Analysis was performed to examine the relative mRNA expression ofvarious MMPs and SerpinE1 in FOCUS-CTL or FOCUS-shFzd2 cells. Resultsare shown in FIG. 13.

Fzd2 Regulates the Phosphorylation Status of Stat3, ERK1/2 and MEK1/2 asWell as its Transcription Activity.

Analysis was performed to examine the phosphorylation of STAT3, ERK1/2,and MEK1/2 in wild-type FOCUS cells, and FOCUS cells with knockdown ofFzd2. Similar analysis was performed in other late stage cell lines andwith treatment with anti-Fzd2 antibody. Results are shown in FIG. 14A.Stat3 transcription activity using a reporter/luciferase-based assay inwild-type FOCUS and SNU449 cells, and cells knockdown with Fzd2 or STAT3is shown in FIG. 14A. Similar results were demonstrated in other latestage cell lines and with treatment with anti-Fzd2 antibody (FIG. 14C).The effect of small molecule inhibitor against Stat3 on cell migrationof late stage HCC cell lines (FOCUS) is shown in FIG. 14C.

Fzd2 Regulates the Phosphorylation Status of Src Family Kinases whichPhosphorylate Stat3 in Late Stage HCC Cell Lines

Phosphorylation of src family kinases was analyzed in wild-type FOCUScells, and FOCUS cells with knockdown of Fzd2 and in other late stagecell lines (FIG. 15A). The effect of inhibiting Src family kinase, withthe small molecule inhibitor (dasatinib), was also examined. Theinhibitor abolished stat3 phosphorylation in FOCUS cells (FIG. 15B). Theeffect of the inhibitor on cell migration of late stage HCC cell lines(FOCUS) was also investigated. Dose-response curves are shown in FIG.15C.

What is claimed:
 1. A method of monitoring antibody mediated Fzd2downmodulation therapy for cancer that exhibits overexpression of Fzd2or overexpression of Wnt5a in a subject, comprising: a) administrationto the subject of an antibody or antigen binding fragment thereof thatspecifically binds Fzd2 within a region corresponding to amino acids125-163 and downmodulates Fzd2, such that the antibody or antigenbinding fragment thereof is delivered to cancer cells of the subject;and b) measuring the expression of one or more of MMP2, MMP3, MMP9, andserpin E1 in the cancer cells before administration and afteradministration wherein reduced expression after administration indicateseffective therapy.
 2. The method of claim 1, further comprisingmeasuring the phosphorylation level of one or more of STAT3, MEK1/2,ERK1/2 and srk family kinases, in the cancer cells before administrationand after administration, wherein reduced phosphorylation afteradministration indicates effective therapy.
 3. The method of claim 1,wherein the antibody promotes internalization of Fzd2 by the cancercells.
 4. The method of claim 1, wherein the antibody prevents ligandbinding to Fzd2.
 5. The method of claim 1, wherein the antibodyspecifically binds to Fzd2 within a region of Fzd2 corresponding toamino acids 134-163 of Fzd2.
 6. The method of claim 1, wherein theantibody specifically binds to Fzd2 within a region of Fzd2corresponding to amino acids 144-163 of Fzd2.
 7. The method of claim 1,wherein the antibody specifically binds to the epitopeHGAEQICVGQNHSEDGAPAL (SEQ ID NO: 1).
 8. The method of claim 1, whereinthe antibody is monoclonal.
 9. The method of claim 1, wherein theantibody is polyclonal.
 10. The method of claim 1, wherein the antibodyis humanized.
 11. The method of claim 1, wherein the cancer is selectedfrom the group consisting of gastrointestinal cancer, prostate cancer,ovarian cancer, breast cancer, head and neck cancer, lung cancer,non-small cell lung cancer, cancer of the nervous system, kidney cancer,retina cancer, skin cancer, liver cancer, pancreatic cancer,genital-urinary cancer and bladder cancer.
 12. The method of claim 11,wherein the cancer is liver cancer.
 13. The method of claim 12, whereinthe cancer is late stage hepatocellular carcinoma.
 14. A method ofmonitoring Fzd2 downmodulation therapy for cancer that exhibitsoverexpression of Fzd2 or overexpression of Wnt5a in a subjectcomprising: a) administration to the subject of an antibody or antigenbinding fragment thereof that specifically binds Fzd2 within a regioncorresponding to amino acids 125-163 and downmodulates Fzd2, such thatthe antibody or antigen binding fragment thereof is delivered to cancercells of the subject; and b) measuring the phosphorylation level of oneor more of STAT3, MEK1/2, ERK1/2 and srk family kinases, in the cancercells before administration and after administration, wherein reducedphosphorylation after administration indicates effective therapy. 15.The method of claim 14, wherein the antibody specifically binds to Fzd2within a region of Fzd2 corresponding to amino acids 134-163 of Fzd2.16. The method of claim 14, wherein the cancer is selected from thegroup consisting of gastrointestinal cancer, prostate cancer, ovariancancer, breast cancer, head and neck cancer, lung cancer, non-small celllung cancer, cancer of the nervous system, kidney cancer, retina cancer,skin cancer, liver cancer, pancreatic cancer, genital-urinary cancer andbladder cancer.
 17. The method of claim 16, wherein the cancer is livercancer.
 18. The method of claim 17, wherein the cancer is late stagehepatocellular carcinoma.
 19. The method of claim 1, wherein the reducedexpression is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100%.
 20. The method of claim 1, wherein measuring expression isby detection of mRNA.
 21. The method of claim 20, wherein detection ofmRNA is by qPCR of mRNA of the tumor using gene specific primers. 22.The method of claim 1, wherein measuring expression is byimmuno-detection of protein.
 23. The method of claim 2, whereinmeasuring the phosphorylation level is by quantitative immunodetectionof the phosphorylated proteins.
 24. The method of claim 14, wherein thereduced phosphorylation is at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100%.
 25. The method of claim 14, wherein measuringthe phosphorylation level is by quantitative immunodetection of thephosphorylated proteins.